U.S. patent application number 13/682589 was filed with the patent office on 2013-11-28 for carboxy x rhodamine analogs.
This patent application is currently assigned to PROMEGA CORPORATION. The applicant listed for this patent is Promega Corporation. Invention is credited to Stephen DWIGHT, Thomas A. KIRKLAND, Mark G. MCDOUGALL.
Application Number | 20130317207 13/682589 |
Document ID | / |
Family ID | 47324436 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130317207 |
Kind Code |
A1 |
KIRKLAND; Thomas A. ; et
al. |
November 28, 2013 |
CARBOXY X RHODAMINE ANALOGS
Abstract
The present invention provides novel fluorescent dyes and kits
containing the same, which are useful for labeling a wide variety
of biomolecules, cells and microorganisms. The present invention
also provides various methods of using the fluorescent dyes for
research and development, forensic identification, environmental
studies, diagnosis, prognosis and/or treatment of disease
conditions.
Inventors: |
KIRKLAND; Thomas A.;
(Atascadero, CA) ; MCDOUGALL; Mark G.; (Arroyo
Grande, CA) ; DWIGHT; Stephen; (Arroyo Grande,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Promega Corporation; |
|
|
US |
|
|
Assignee: |
PROMEGA CORPORATION
Madison
WI
|
Family ID: |
47324436 |
Appl. No.: |
13/682589 |
Filed: |
November 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61562021 |
Nov 21, 2011 |
|
|
|
Current U.S.
Class: |
536/26.8 ;
536/28.54; 540/578; 540/581 |
Current CPC
Class: |
C07D 491/22 20130101;
C07H 19/10 20130101; G01N 2333/90241 20130101; C07D 491/147
20130101; C07F 9/65586 20130101; C07H 19/073 20130101; C07D 223/16
20130101; C07D 471/06 20130101; C12Q 1/686 20130101; G01N 33/542
20130101; G01N 33/6845 20130101; G01N 33/582 20130101; C07D 491/16
20130101; C09B 11/24 20130101 |
Class at
Publication: |
536/26.8 ;
540/578; 540/581; 536/28.54 |
International
Class: |
C07H 19/10 20060101
C07H019/10; C07H 19/073 20060101 C07H019/073; C07D 491/22 20060101
C07D491/22; C07D 471/06 20060101 C07D471/06; C07D 491/147 20060101
C07D491/147; C07D 491/16 20060101 C07D491/16 |
Claims
1. A compound according to claim 8, wherein the compound is a
compound of formula (Ia) or (Ib): ##STR00122## wherein R.sup.1 and
R.sup.11 are independently H or C.sub.1-4 alkyl, L-R or L-C.sub.S;
L is a covalent linkage that is linear or branched, cyclic or
heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond; R is a
reactive group; C.sub.S is a conjugated substance; R.sup.2,
R.sup.5, R.sup.12 and R.sup.15 are independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S; R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24,
R.sup.25, and R.sup.26 are independently H or C.sub.1-6 alkyl or
one or more of R.sup.20 and R.sup.21, R.sup.21 and R.sup.22,
R.sup.22 and R.sup.23, R.sup.24 and R.sup.25, R.sup.25 and
R.sup.26, and R.sup.26 and R.sup.23 together form an aryl,
heteroaryl, carbocyclic or heterocyclic ring; R.sup.1 and R.sup.2
and/or R.sup.11 and R.sup.12 may together form a carbocyclic,
heterocyclic, aryl or heteroaryl ring; R.sup.6-10 are independently
H, halo, OH, alkyl, aryl, heteroaryl, CO.sub.2H, SO.sub.3H,
L-CO.sub.2H, L-SO.sub.3H, L-R or L-C.sub.S; each X is independently
CHR.sup.23, O, S or NR.sup.30; and R.sup.30 is H, C.sub.1-4 alkyl
or --C(O)C.sub.1-4 alkyl.
2.-4. (canceled)
5. A compound according to claim 8 wherein at least one of
R.sup.24, R.sup.25 or R.sup.26 is H.
6.-7. (canceled)
8. A compound according to formula (IIIa), (IIIb) or (IIIc):
##STR00123## wherein R.sup.11 is independently H or C.sub.1-4
alkyl, L-R or L-C.sub.S; L is a covalent linkage that is linear or
branched, cyclic or heterocyclic saturated or unsaturated, having
1-16 non hydrogen atoms such that the linkage contains any
combination of ester, acid, amine, amide, alcohol, ether, thioether
or halide groups or single, double, triple or aromatic
carbon-carbon bond; R is a reactive group; C.sub.S is a conjugated
substance; R.sup.2 and R.sup.16 can be independently H, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S; R.sup.3 and R.sup.4 are H, alkyl, L-R, L-C.sub.S,
L-CO.sub.2H, L-SO.sub.3H or together form a carbocyclic, aryl,
heteroaryl, or heterocyclic ring; alternatively, R.sup.2 and
R.sup.3 and independently R.sup.4 and R.sup.16 together form a
carbocyclic, heterocyclic, aryl or heteroaryl ring; R.sup.5,
R.sup.12, R.sup.13, R.sup.14 and R.sup.15 are independently H,
alkyl, aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H,
L-SO.sub.3H, L-R or L-C.sub.S; R.sup.20, R.sup.21, R.sup.22 and
R.sup.23 are independently H or C.sub.1-6 alkyl or one or more of
R.sup.20 and R.sup.21, R.sup.21 and R.sup.22, R.sup.22 and
R.sup.23, together form an aryl, heteroaryl, carbocyclic or
heterocyclic ring; R.sup.11 and R.sup.12 may together form a
carbocyclic, heterocyclic, aryl or heteroaryl ring; R.sup.6-10 are
independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl,
CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or L-C.sub.S; X
is CHR.sup.23, O, S or NR.sup.30; and R.sup.30 is H, C.sub.1-4
alkyl or --C(O)C.sub.1-4 alkyl.
9. A compound according to claim 8 wherein X is CH.sub.2.
10.-11. (canceled)
12. A compound according to claim 8 wherein R.sup.1 and R.sup.2
form a 5-7 membered carbocyclic ring.
13.-15. (canceled)
16. A compound according to claim 8 wherein R.sup.12 is H, Cl or
OMe.
17. A compound according to claim 8 wherein R.sup.11 and R.sup.12
form a 5-7 membered carbocyclic ring.
18. (canceled)
19. A compound according to claim 8 wherein at least one of
R.sup.20, R.sup.21, R.sup.22 and R.sup.23 is H.
20.-24. (canceled)
25. A compound according to claim 8 wherein R.sup.2 is H, Cl or
OMe.
26. A compound according to claim 8 wherein R.sup.3 is C.sub.1-4
alkyl.
27. A compound according to claim 26 wherein R.sup.3 is methyl or
ethyl.
28. A compound according to claim 8 any one of claims 2-27 wherein
R.sup.4 is C.sub.1-4 alkyl.
29. A compound according to claim 28 wherein R.sup.4 is methyl or
ethyl.
30. A compound according to claim 8 wherein R.sup.3 is part of a
heterocycle.
31. (canceled)
32. A compound according to claim 8 wherein R.sup.4 is part of a
heterocycle.
33.-44. (canceled)
45. A compound according to claim 8 wherein R.sup.5 is H.
46. A compound according to claim 8 wherein R.sup.15 is H.
47. A compound according to claim 8 wherein R.sup.6 is H or
halogen.
48. A compound according to claim 8 wherein R.sup.9 is H or
halogen.
49. A compound according to claim 8 wherein R.sup.10 is H, F, Cl
CO.sub.2H or SO.sub.2H.
50. A compound according to claim 8 wherein one of R.sub.7 and
R.sub.8 is -L-R, -L-CO.sub.2H or -L-C.sub.S and the other is H, Cl,
or F.
51. A compound according to claim 8 wherein L is --CO--,
--SCH.sub.2CO--, or --SO.sub.2--.
52. A compound according to claim 8 is a self-immolative
linker.
53. A compound according to claim 8 wherein R is ##STR00124##
54. A compound according to claim 8 wherein C.sub.S is
NHCH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.n(CH.sub.2).sub.6Cl,
wherein n is 2-6.
55. A compound according to claim 8 wherein C.sub.S comprises a
nucleoside.
56. A compound according to claim 8 wherein C.sub.S comprises an
oligonucleotide.
57.-82. (canceled)
83. A compound according to formula (IXa) or (IXb): ##STR00125##
wherein R.sup.11 is H or C.sub.1-4 alkyl, L-R or L-C.sub.S; L is a
covalent linkage that is linear or branched, cyclic or heterocyclic
saturated or unsaturated, having 1-16 non hydrogen atoms such that
the linkage contains any combination of ester, acid, amine, amide,
alcohol, ether, thioether or halide groups or single, double,
triple or aromatic carbon-carbon bond; R is a reactive group;
C.sub.S is a conjugated substance; R.sup.12 and R.sup.15 are
independently H, alkyl, aryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H,
L-SO.sub.3H, L-R or L-C.sub.S; R.sup.20, R.sup.21, R.sup.22 and
R.sup.23 are independently H or C.sub.1-6 alkyl or one or more of
R.sup.20 and R.sup.21, R.sup.21 and R.sup.22 and R.sup.22 and
R.sup.23 together form a fused aryl ring; R.sup.11 and R.sup.12 may
be joined together in an optionally substituted ring; R.sup.6-10
are independently H, F, Cl, Br, I, OH, alkyl, aryl, CO.sub.2H,
SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or L-C.sub.S; X is
CHR.sup.23, O, S or NR.sup.30; and R.sup.30 is H, C.sub.1-4 alkyl
or --C(O)C.sub.1-4 alkyl.
84. A compound selected from the group consisting of: ##STR00126##
##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131##
##STR00132## ##STR00133## ##STR00134##
Description
FIELD OF THE INVENTION
[0001] The invention relates to fluorescent dyes and methods of
using them.
BACKGROUND
[0002] Fluorescent dyes are widely used in biological research and
medical diagnostics. Fluorescent dyes tend to be superior to
conventional techniques because they are less expensive, less toxic
and can generally be detected with sufficient sensitivity. A
diversity of fluorescent dyes with a distinguishable color range
has made it more practical to perform multiplexed assays capable of
detecting multiple biologic targets at the same time.
[0003] Further improvement in the properties of the dyes is needed
in order to meet the increasing demands of new instruments and new
biological applications. In particular, additional strategies to
allow for fine-tuning of the wave-lengths of the dyes for maximal
signal detection and to provide additional colors are needed.
SUMMARY
[0004] In one aspect, the invention provides a compound according
to formula (Ia) or (Ib):
##STR00001##
wherein
[0005] R.sup.1 and R.sup.11 are independently H or C.sub.1-4 alkyl,
L-R or L-C.sub.S;
[0006] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0007] R is a reactive group;
[0008] C.sub.S is a conjugated substance;
[0009] R.sup.2, R.sup.5, R.sup.12 and R.sup.15 are independently H,
alkyl, aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H,
L-SO.sub.3H, L-R or L-C.sub.S;
[0010] R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25,
and R.sup.26 are independently H or C.sub.1-6 alkyl or one or more
of R.sup.20 and R.sup.21, R.sup.21 and R.sup.22, R.sup.22 and
R.sup.23, R.sup.24 and R.sup.25, R.sup.25 and R.sup.26, and
R.sup.26 and R.sup.23 together form an aryl, heteroaryl,
carbocyclic or heterocyclic ring;
[0011] R.sup.1 and R.sup.2 and/or R.sup.11 and R.sup.12 may
together form a carbocyclic, heterocyclic, aryl or heteroaryl
ring;
[0012] R.sup.6-10 are independently H, halo, OH, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0013] each X is independently CHR.sup.23, O, S or NR.sup.30;
and
[0014] R.sup.30 is H, C.sub.1-4 alkyl or --C(O)C.sub.1-4 alkyl.
[0015] In another aspect, the invention provides a compound
according to formula (IIa) or (IIb):
##STR00002##
wherein
[0016] R.sup.1 and R.sup.11 are independently H or C.sub.1-4 alkyl,
L-R or L-C.sub.S;
[0017] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0018] R is a reactive group;
[0019] C.sub.S is a conjugated substance;
[0020] R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.12, R.sup.13,
R.sup.14 and R.sup.15 are independently H, alkyl, aryl, heteroaryl,
CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0021] R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25
and R.sup.26 are independently H or C.sub.1-6 alkyl or one or more
of R.sup.20 and R.sup.21, R.sup.21 and R.sup.22, R.sup.22 and
R.sup.23, R.sup.24 and R.sup.25, R.sup.25 and R.sup.26, and
R.sup.26 and R.sup.23 together form an aryl, heteroaryl,
carbocyclic or heterocyclic ring;
[0022] R.sup.1 and R.sup.2 and/or R.sup.11 and R.sup.12 may
together form a carbocyclic, heterocyclic, aryl or heteroaryl
ring;
[0023] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S;
[0024] each X is independently CHR.sup.23, O, S or NR.sup.30;
and
[0025] R.sup.30 is H, C.sub.1-4 alkyl or --C(O)C.sub.1-4 alkyl.
[0026] In a further aspect, the invention provides a compound
according to formula (IIIa), (IIIb) or (IIIc):
##STR00003##
wherein
[0027] R.sup.11 is independently H or C.sub.1-4 alkyl, L-R or
L-C.sub.S;
[0028] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0029] R is a reactive group;
[0030] C.sub.S is a conjugated substance;
[0031] R.sup.2 and R.sup.16 can be independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0032] R.sup.3 and R.sup.4 are H, alkyl, L-R, L-C.sub.S,
L-CO.sub.2H, L-SO.sub.3H or together form a carbocyclic, aryl,
heteroaryl, or heterocyclic ring;
[0033] alternatively, R.sup.2 and R.sup.3 and independently R.sup.4
and R.sup.16 together form a carbocyclic, heterocyclic, aryl or
heteroaryl ring;
[0034] R.sup.5, R.sup.12, R.sup.13, R.sup.14 and R.sup.15 are
independently H, alkyl, aryl, heteroaryl, CO.sub.2H, SO.sub.3H,
L-CO.sub.2H, L-SO.sub.3H, L-R or L-C.sub.S;
[0035] R.sup.20, R.sup.21, R.sup.22 and R.sup.23 are independently
H or C.sub.1-6 alkyl or one or more of R.sup.20 and R.sup.21,
R.sup.21 and R.sup.22, R.sup.22 and R.sup.23, together form an
aryl, heteroaryl, carbocyclic or heterocyclic ring;
[0036] R.sup.11 and R.sup.12 may together form a carbocyclic,
heterocyclic, aryl or heteroaryl ring;
[0037] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S;
[0038] X is CHR.sup.23, O, S or NR.sup.30; and
[0039] R.sup.30 is H, C.sub.1-4 alkyl or --C(O)C.sub.1-4 alkyl.
[0040] In yet another aspect, the invention provides a compound
according to formula (IV):
##STR00004##
wherein
[0041] R.sup.2 and R.sup.16 can be independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0042] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0043] R is a reactive group;
[0044] C.sub.S is a conjugated substance;
[0045] R.sup.3 and R.sup.4 are H, alkyl or together form a
carbocyclic, aryl, heteroaryl, or heterocyclic ring;
[0046] alternatively, R.sup.2 and R.sup.3 and independently R.sup.4
and R.sup.16 together form a carbocyclic, heterocyclic, aryl or
heteroaryl ring;
[0047] R.sup.5 and R.sup.15 are independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0048] R.sup.22 and R.sup.23 are independently H or C.sub.1-6 alkyl
or together form an aryl, heteroaryl, carbocyclic or heterocyclic
ring; and
[0049] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S.
[0050] In a further aspect, the invention provides a compound
according to formula (V):
##STR00005##
wherein
[0051] R.sup.2 and R.sup.16 can be independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0052] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0053] R is a reactive group;
[0054] C.sub.S is a conjugated substance;
[0055] R.sup.3 and R.sup.4 are H, alkyl, or together form a
carbocyclic, heterocyclic, aryl or heteroaryl ring;
[0056] alternatively, R.sup.2 and R.sup.3 and independently R.sup.4
and R.sup.16 may together form a carbocyclic, heterocylic, aryl or
heteroaryl ring;
[0057] R.sup.5 and R.sup.15 are independently H, alkyl, aryl,
heteoaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0058] R.sup.20 and R.sup.21 are independently H or C.sub.1-6 alkyl
or together form an aryl, heteroaryl, carbocyclic or heterocyclic
ring; and
[0059] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S.
[0060] In another aspect, the invention provides a compound
according to formula (VI):
##STR00006##
wherein
[0061] R.sup.5 and R.sup.15 are independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0062] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0063] R is a reactive group;
[0064] C.sub.S is a conjugated substance;
[0065] R.sup.22, R.sup.23, R.sup.26 and R.sup.27 are independently
H or C.sub.1-6 alkyl or one or more of R.sup.22 and R.sup.23 and
R.sup.26 and R.sup.27 together form an aryl, heteroaryl,
carbocyclic or heterocyclic ring; and
[0066] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S.
[0067] In an additional aspect, the invention provides a compound
according to formula (VII):
##STR00007##
wherein
[0068] R.sup.5 and R.sup.15 are independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0069] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0070] R is a reactive group;
[0071] C.sub.S is a conjugated substance;
[0072] R.sup.20, R.sup.21, R.sup.24 and R.sup.25 are independently
H or C.sub.1-6 alkyl or one or more of R.sup.20 and R.sup.21 and
R.sup.24 and R.sup.25 together form an aryl, heteroaryl,
carbocyclic or heterocyclic ring; and
[0073] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S.
[0074] In a further aspect, the invention provides a compound of
formula (VIIIa) or (VIIIb):
##STR00008##
wherein
[0075] R.sup.11 is H or C.sub.1-4 alkyl, L-R or L-C.sub.S;
[0076] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0077] R is a reactive group;
[0078] C.sub.S is a conjugated substance;
[0079] R.sup.12 and R.sup.15 are independently H, alkyl, aryl,
CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0080] R.sup.20, R.sup.21, R.sup.22 and R.sup.23 are independently
H or C.sub.1-6 alkyl or one or more of R.sup.20 and R.sup.21,
R.sup.21 and R.sup.22 and R.sup.22 and R.sup.23 together form a
fused aryl ring;
[0081] R.sup.11 and R.sup.12 may be joined together in an
optionally substituted ring;
[0082] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0083] X is CHR.sup.23, O, S or NR.sup.30; and
[0084] R.sup.30 is H, C.sub.1-4 alkyl or --C(O)C.sub.1-4 alkyl.
[0085] In some aspects, the invention provides a labeled
biomolecule. In some aspects, the labeled biomolecule is a small
molecule, e.g., a drug or drug compound. In other aspects, the
labeled biomolecule or labeled small molecule acts as a fluorescent
tracer to monitor binding to a target, e.g., drug target.
[0086] In other aspects, the invention provides a method to detect
a selected molecule in a sample, comprising: a) contacting a sample
suspected of containing a selected molecule with a composition
comprising a conjugate comprising a compound according to the
present invention and a ligand for the selected molecule so as to
yield a mixture; and b) detecting the presence or amount of the
compound in the mixture.
[0087] In some aspects, the invention provides a method of
detecting the presence of a nucleic acid polymer in a sample
comprising: contacting a sample suspected of containing a nucleic
acid polymer with a composition comprising a conjugate comprising a
compound according to the present invention and an oligonucleotide;
and detecting the presence or amount of the compound in the
sample.
[0088] In some aspects, the invention provides a method of
monitoring binding to a target of interest, e.g., a drug target,
comprising contacting a sample comprising a fusion protein
comprising the target of interest with a small molecule conjugated
to a dye described herein; and detecting binding of the small
molecule conjugate to the target of interest. In some aspects, the
fusion protein comprises a luciferase protein fused to the target
of interest. In other aspects, the fusion protein comprises a
fluorescent protein fused to the target of interest. In some
aspects, wherein the fusion protein comprises a luciferase protein,
binding is detected by bioluminescence resonance energy transfer
(BRET). In some aspects, wherein the fusion protein comprises a
fluorescent protein, binding is detected by fluorescent resonance
energy transfer (FRET). In some aspects, the sample comprises a
cell expressing the fusion protein.
[0089] In some aspects, the invention provides a method for
monitoring protein-protein interactions comprising contacting a
sample comprising a first fusion protein and a second fusion
protein with a ligand conjugated to a dye described herein; and
detecting the interaction between the first and second fusion
protein. In some aspects, the first fusion protein comprises a
luciferase protein fused to a first binding partner, the second
fusion protein comprises a HaloTag.RTM. protein fused to a second
binding partner, and the ligand conjugate comprises a HaloTag.RTM.
ligand.
[0090] In some aspects, the invention provides reactive dyes that
may be used to label a protein(s), peptide(s) or ligand(s). In some
aspects, the reactive dyes of the invention could be attached to a
target protein or peptide using a reactive cyanobenzothiazole
labeling chemistry.
[0091] In other aspects, the invention provides a kit comprising a
compound according to the present invention or a labeled
biomolecule according to the present invention. In some aspects,
the kit comprises a labeled biomolecule or labeled small molecule,
e.g., drug or drug compound. In some aspects, the kit further
comprises cells expressing a fusion protein comprising a protein or
target of interest. In other aspects, the kit further comprises a
vector for expressing a fusion protein comprising a protein or
target of interest.
BRIEF DESCRIPTION OF THE DRAWINGS
[0092] FIG. 1 shows various compounds according to the present
invention.
[0093] FIG. 2 shows various compounds according to the present
invention.
[0094] FIG. 3 shows various compounds according to the present
invention.
[0095] FIG. 4 shows electropherograms showing peaks of loci labeled
with FAM, JOE, ET TMR, ET ROX and ET 4510. Male DNA (1.0 ng) was
amplified using a 21-STR Primer Pair Mix (Table 1) and the
additional loci D22S1045, D2S441 and DYS391. Amplification products
were analyzed with an Applied Biosystems 3500 xL Genetic Analyzer.
Panel A. An electropherogram showing the peaks of the FAM-labeled
loci: Amelogenin, D3S1358, D1S1656, D6S1043, D13S317 and Penta E.
Panel B. An electropherogram showing the peaks of the JOE-labeled
loci: Penta D, D16S539, D18S51, D2S1338 and CSF1PO. Panel C. An
electropherogram showing the peaks of the ET TMR-labeled loci:
TH01, vWA, D21S11, D7S820, D5S818, and TPDX. Panel D. An
electropherogram showing ET ROX-labeled loci: D8S1179, D12S391,
D19S433 and FGA. Panel E. An electropherogram showing ET
4510-labeled loci: D22S1045, D2S441 and DYS391. Note: Penta D is
the largest loci in the JOE-labeled loci set, and the ET
4510-labeled loci are shown from smallest to largest. Loci in the
other sets are shown from largest to smallest.
[0096] FIG. 5 shows electropherograms showing peaks of the loci
DYS391, D2S441 and D22S 1045 labeled with 4510 (Panel A), 4563
(Panel B) or 4574 (Panel C). Male DNA (1.0 ng) was amplified using
a 21-STR Primer Pair Mix (Table 1) and the additional loci
D22S1045, D2S441 and DYS391. Amplification products were analyzed
with an Applied Biosystems 3500 xL Genetic Analyzer.
[0097] FIG. 6 shows electropherograms showing peaks of the loci
DYS391, D2S441 and D22S 1045 labeled with 4510 (Panel A), 4563
(Panel B), or 4574 (Panel C) and the JOE labeled loci: Penta D,
D16S539, D18S51, D2S1338 and CSF1PO. Male DNA (1.0 ng) was
amplified using a 21-STR Primer Pair Mix (Table 1) and the
additional loci D22S1045, D2S441 and DYS391. Amplification products
were analyzed with an Applied Biosystems 3500 xL Genetic
Analyzer.
[0098] FIG. 7 shows electropherograms showing peaks of the loci
DYS391, D2S441 and D22S 1045 labeled with 4510 (Panel A), 4563
(Panel B), or 4574 (Panel C). Male DNA (0.5 ng) was amplified using
a 21-STR Primer Pair Mix (Table 1) and the additional loci
D22S1045, D2S441 and DYS391. Amplification products were analyzed
with an Applied Biosystems 3500 xL Genetic Analyzer.
[0099] FIG. 8 shows electropherograms showing peaks of the loci
DYS391, D2S441 and D22S 1045 labeled with 4510 (Panel A), 4563
(Panel B), or 4574 (Panel C) and the JOE labeled loci: Penta D,
D16S539, D18S51, D2S1338 and CSF1PO. Male DNA (0.5 ng) was
amplified using a 21-STR Primer Pair Mix (Table 1) and the
additional loci D22S1045, D2S441 and DYS391. Amplification products
were analyzed with an Applied Biosystems 3500 xL Genetic
Analyzer.
[0100] FIG. 9 shows confocal images using ligand 3780. (a) U2OS
cells stably expressing HaloTag.RTM. containing a nuclear
localization sequence (HT-NLS) were labeled with 100 nM ligand 3780
by a no-wash protocol and imaged using 3% .lamda.633 laser, PMT
715, CA 80 .mu.m, 100.times.. (b) U2OS cells stably expressing
HT-NLS were labeled with 1 .mu.M ligand 3780 by a rapid label
protocol and imaged using 3% .lamda.633 laser, PMT 600, CA 200
.mu.m, 100.times.. (c) U2OS cells stably expressing the fusion
protein p65-HaloTag (p65-HT) were labeled with 104 ligand 3780 by a
rapid label protocol and imaged using 8% .lamda.633 laser, PMT 775,
CA 80 .mu.m, 100.times.. (d) U2OS cells labeled with 1 .mu.M ligand
3780 by a rapid label protocol and imaged using 8% .lamda.633
laser, PMT 775, CA 200 .mu.m, 20.times.. The left panel in each
image shows fluorescence channel, the middle panel shows DIC, and
the right panel shows an overlay of the two.
[0101] FIG. 10 shows confocal images using ligand 3781. (a) U2OS
cells stably expressing the fusion protein p65-HT and (b) U2OS
cells were labeled with 1 .mu.M ligand 3781 by a rapid label
protocol. Cells were imaged using 10% .lamda.543 laser, PMT 830, CA
80 .mu.m, 80.times.. The left panel in each image shows
fluorescence channel, the middle panel shows DIC, and the right
panel shows an overlay of the two.
[0102] FIG. 11 shows confocal images using ligand 3782. (a) U2OS
cells stably expressing HT-NLS were labeled with 100 nM ligand 3782
by a no-wash protocol and imaged using 3% .lamda.633 laser, PMT
615, CA 80 .mu.m, 100.times.. (b) U2OS cells stably expressing
HT-NLS were labeled with 1 .mu.M ligand 3782 by a rapid label
protocol and imaged using 3% .lamda.633 laser, PMT 600, CA 200
.mu.m, 100.times.. (c) U2OS stably expressing the fusion protein
p65-HT were labeled with 1 .mu.M ligand 3782 by a rapid label
protocol and imaged using 8% .lamda.633 laser, PMT 775, CA 80
.mu.m, 100.times.. (d) U2OS cells were labeled with 1 .mu.M ligand
3782 by a rapid label protocol and imaged using 8% .lamda.633
laser, PMT 775, CA 200 .mu.m, 20.times.. The left panel in each
image shows fluorescence channel, the middle panel shows DIC, and
the right panel shows an overlay of the two.
[0103] FIG. 12 shows confocal images using ligand 3783. (a) U2OS
cells stably expressing HT-NLS were labeled with 100 nM ligand 3783
by a no-wash protocol and imaged using 3% .lamda.633 laser, PMT
615, CA 80 .mu.m, 100.times.. (b) U2OS cells stably expressing
HT-NLS were labeled with 1 .mu.M ligand 3783 by a rapid label
protocol and imaged using 3% .lamda.633 laser, PMT 600, CA 200
.mu.m, 100.times.. (c) U2OS cells stably expressing the fusion
protein p65-HT were labeled with 1 .mu.M ligand 3783 by the rapid
label protocol and imaged using 8% .lamda.633 laser, PMT 750, CA 80
.mu.m, 100.times.. (d) U2OS cells were labeled with 1 .mu.M ligand
3783 by a rapid labeled protocol and imaged using 8% .lamda.633
laser, PMT 775, CA 200 .mu.m, 20.times.. The left panel in each
image shows fluorescence channel, the middle panel shows DIC, and
the right panel shows an overlay of the two.
[0104] FIG. 13 shows confocal images using ligand 3905. (a) U2OS
cells stably expressing HT-NLS were labeled with 1 .mu.M ligand
3905 by a rapid label protocol and imaged using 3% .lamda.633
laser, PMT 600, CA 200 .mu.m, 20.times.. (b) U2OS cells stably
expressing the fusion protein p65-HT and (c) U2OS cells were
labeled with 1 .mu.M ligand 3905 by a rapid label protocol and
imaged using 15% .lamda.633 laser, PMT800, CA 80 .mu.m, 20.times..
The left panel in each image shows fluorescence channel, the middle
panel shows DIC, and the right panel shows an overlay of the
two.
[0105] FIG. 14 shows confocal images using ligand 3906. (a) U2OS
cells stably expressing HT-NLS were labeled with 1 .mu.M ligand
3906 by a rapid label protocol and imaged using 3% .lamda.633
laser, PMT 600, CA 200 .mu.m, 20.times.. (b) U2OS cells stably
expressing the fusion protein p65-HT and (c) U2OS cells were
labeled with 1 .mu.M ligand 3906 by a rapid label protocol and
imaged using 10% .lamda.633 laser, PMT 720, CA 80 .mu.m, 20.times..
The left panel in each image shows fluorescence channel, the middle
panel shows DIC, and the right panel shows an overlay of the
two.
[0106] FIG. 15 shows confocal images of ligand 3954. (a) U2OS cells
stably expressing HT-NLS and (b) U2OS cells were labeled with 1
.mu.M ligand 3954 by a rapid label protocol and imaged using 4%
.lamda.633 laser, PMT 880, CA 80 .mu.m, 20.times.. The left panel
in each image shows fluorescence channel, the middle panel shows
DIC, and the right panel shows an overlay of the two.
[0107] FIG. 16 shows confocal images of ligand 4356 and 4357. U2OS
cells stably expressing HT-NLS were labeled with 1 .mu.M of ligand
(a) 4356 or (b) 4357 by a rapid label protocol and imaged using 10%
.lamda.633 laser, PMT 830, CA 80 .mu.m, 30.times.. The left panel
in each images shows fluorescence channel, the middle panel shows
DIC, and the right panel shows an overlay of the two.
[0108] FIG. 17 shows the labeling efficiency of ligands 3780, 3782
and 3783 in U2OS cells stably expressing HT-NLS. (a) Fluorescent
scans after SDS-PAGE showing ligand signal directly (.lamda.633)
and TMR signal (.lamda.532). Lane 1 represents 100 nM ligand using
a no-wash labeling (pulse) with 5 .mu.M TMR rapid labeling (chase),
lane 2 represents 1 .mu.M rapid labeling (pulse) with 5 .mu.M TMR
rapid labeling (chase), lane 3 represents 5 .mu.M rapid labeling
(pulse) with 5 .mu.M TMR rapid labeling (chase), "TMR" represents
cells labeled only with TMR ligand. (b) Graph showing
quantification of bands (TMR signal) from TMR gel as a percent of
TMR alone band.
[0109] FIG. 18 shows viability of U2OS and CHO cells in the
presence of ligand 3782. Graph shows results of CellTiter-Glo.RTM.
Luminescent Cell Viability Assay after 24 hour incubation with
ligand 3782 or DMSO carrier (control). Each bar represents n=6
wells, .+-.SEM.
[0110] FIG. 19 shows the performance of ligand 3782 in a gel-based
analysis as an alternative to the HaloTag.RTM. TMR ligand.
[0111] FIG. 20 shows a schematic representation of a p38alpha
kinase fluorescent tracer PBI 4838 binding to an inactive p38alpha
kinase.
[0112] FIG. 21 shows a titration of the dye tracer PBI 4838 with
cells expressing a NanoLuc-p38 fusion protein monitored using BRET.
In addition, the figure shows that the interaction of the tracer
and NanoLuc-p38 fusion can be inhibited by BIRB796. Binding was
monitored using BRET in lysed cells (A) and live cells (B).
[0113] FIG. 22 shows monitoring of the binding of a known drug,
BIRB796, to a kinase target, p38, in living cells using a
fluorescent dye tracer PBI 4838. Binding was monitored using BRET
and the EC50 value determined for p38 (A) and PKCa (negative
control) (B).
[0114] FIG. 23 shows a rapamycin-dependent increase of BRET
occurring between a NanoLuc-Frb fusion (donor) and a HaloTag-FKBP12
fusion bound to PBI 3781, a dye conjugated HaloTag.RTM. ligand (A).
(B) shows the emission spectra for NanoLuc luciferase and PBI 3781
and excitation spectra for PBI 3781
[0115] FIG. 24 shows the monitoring of the rapamycin-mediated
interaction between a NanoLuc-Frb fusion and HaloTag-FKPf12 fusion
using BRET and a dye conjugated HaloTag.RTM. ligand (PBI 3781).
DETAILED DESCRIPTION
Definitions
[0116] As used herein, the following terms and expressions have the
indicated meanings Specific values listed below for radicals,
substituents, and ranges are for illustration only. They do not
exclude other defined values or other values within defined ranges
for the radicals and substituents.
[0117] When a group or moiety can be substituted, the term
"substituted" indicates that one or more (e.g., 1, 2, 3, 4, 5, or
6; in some embodiments 1, 2, or 3; and in other embodiments 1 or 2)
hydrogens on the group indicated in the expression using
"substituted" can be replaced with a selection of recited indicated
groups or with a suitable group known to those of skill in the art
(e.g., one or more of the groups recited below), provided that the
indicated atom's normal valency is not exceeded, and that the
substitution results in a stable compound. Suitable substituents of
a substituted group can include alkyl, alkenyl, alkynyl, alkoxy,
halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl,
heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino,
alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl,
acetylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy,
carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl,
alkylsulfonyl, arylsulfinyl, arylsulfonyl, heteroarylsulfinyl,
heteroarylsulfonyl, heterocyclesulfinyl, heterocyclesulfonyl,
phosphate, sulfate, hydroxylamine, hydroxyl (alkyl)amine, and
cyano. Additionally, the suitable indicated groups can include,
e.g., --X, --R, --O--, --OR, --SR, --S--, --NR.sub.2, --NR.sub.3,
.dbd.NR, --CX.sub.3, --CN, --OCN, --SCN, --N.dbd.C.dbd.O, --NCS,
--NO, --NO.sub.2, .dbd.N.sub.2, --N.sub.3, NC(.dbd.O)R,
--C(.dbd.O)R, --C(.dbd.O)NRR --S(.dbd.O).sub.2O--,
--S(.dbd.O).sub.2OH, --S(.dbd.O).sub.2R, --OS(.dbd.O).sub.2OR,
--S(.dbd.O).sub.2NR, --S(.dbd.O)R, --OP(.dbd.O)O.sub.2RR,
--P(.dbd.O)O.sub.2RR, --P(.dbd.O)(O--).sub.2,
--P(.dbd.O)(OH).sub.2, --C(.dbd.O)R, --C(.dbd.O)X, --C(S)R,
--C(O)OR, --C(O)O--, --C(S)OR, --C(O)SR, --C(S)SR, --C(O)NRR,
--C(S)NRR, --C(NR)NRR, where each X is independently a halogen
("halo"): F, Cl, Br, or I; and each R is independently H, alkyl,
aryl, heteroaryl, heterocycle, a protecting group or prodrug
moiety. As would be readily understood by one skilled in the art,
when a substituent is keto (.dbd.O) or thioxo (.dbd.S), or the
like, then two hydrogen atoms on the substituted atom are
replaced.
[0118] As used herein, the term "alkyl" refers to a branched,
unbranched, or cyclic hydrocarbon having, for example, from 1 to 20
carbon atoms, and often 1 to 12, or 1 to about 6 carbon atoms.
Examples include, but are not limited to, methyl, ethyl, 1-propyl,
2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl
(t-butyl), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl,
3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl,
2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,
4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,
2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, hexyl, octyl, decyl,
dodecyl, and the like. The alkyl can be unsubstituted or
substituted. For example, a substituted alkyl group can be a
haloalkyl group, as described below. The alkyl can also be
optionally partially or fully unsaturated. As such, the recitation
of an alkyl group includes both alkenyl and alkynyl groups. The
alkyl can be a monovalent hydrocarbon radical, as described and
exemplified above, or it can be a divalent hydrocarbon radical
(i.e., alkylene), according to the context of its usage.
Additionally, the alkyl group can be optionally interrupted, as
described below for the term interrupted.
[0119] The term "alkenyl" refers to a monoradical branched or
unbranched partially unsaturated hydrocarbon chain (i.e. a
carbon-carbon, sp.sup.2 double bond). In one embodiment, an alkenyl
group can have from 2 to 10 carbon atoms, or 2 to 6 carbon atoms.
In another embodiment, the alkenyl group has from 2 to 4 carbon
atoms. Examples include, but are not limited to, ethylene or vinyl,
allyl, cyclopentenyl, 5-hexenyl, and the like. The alkenyl can be
unsubstituted or substituted.
[0120] The term "alkynyl" refers to a monoradical branched or
unbranched hydrocarbon chain, having a point of complete
unsaturation (i.e. a carbon-carbon, sp triple bond). In one
embodiment, the alkynyl group can have from 2 to 10 carbon atoms,
or 2 to 6 carbon atoms. In another embodiment, the alkynyl group
can have from 2 to 4 carbon atoms. This term is exemplified by
groups such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,
2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1-octynyl,
and the like. The alkynyl can be unsubstituted or substituted.
[0121] The term "cycloalkyl" or "carbocyle" or "carbocyclic" refers
to cyclic alkyl groups of from 3 to about 10 carbon atoms having a
single cyclic ring or multiple condensed rings. Such cycloalkyl
groups include, by way of example, single ring structures such as
cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or
multiple ring structures such as adamantanyl, and the like. The
cycloalkyl can be unsubstituted or substituted. The cycloalkyl
group can be monovalent or divalent, and can be optionally
substituted as described above for alkyl groups. The cycloalkyl
group can optionally include one or more cites of unsaturation, for
example, the cycloalkyl group can include one or more carbon-carbon
double bonds, such as, for example, cyclohexene,
1,3-cyclohexadiene, 1,4-cyclohexadiene, and the like. The
cycloalkyl group can be a carbocycle, which refers to a saturated
or partially unsaturated ring having 3 to 8 carbon atoms as a
monocycle, 7 to 12 carbon atoms as a bicycle, and up to about 20
carbon atoms as a polycycle. Monocyclic carbocycles typically have
3 to 6 ring atoms, still more typically 5 or 6 ring atoms. Bicyclic
carbocycles have 7 to 12 ring atoms, e.g., arranged as a
bicyclo[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms
arranged as a bicyclo[5,6] or [6,6] system. Examples of carbocycles
include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl,
1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,
1-cyclohex-1-enyl, 1-cyclohex-2-enyl, or 1-cyclohex-3-enyl. The
carbocycle can be optionally substituted as described above for
alkyl groups.
[0122] The term "alkoxy" refers to the group alkyl-O--, where alkyl
is as defined herein.
[0123] In one embodiment, alkoxy groups include, e.g., methoxy,
ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,
n-pentoxy, n-hexyloxy, 1,2-dimethylbutoxy, and the like. The alkoxy
can be unsubstituted or substituted.
[0124] The term "aryl" refers to an aromatic hydrocarbon group
derived from the removal of one hydrogen atom from a single carbon
atom of a parent aromatic ring system. The radical can be at a
saturated or unsaturated carbon atom of the parent ring system. The
aryl group can have 6-18 carbon atoms, 6-14 carbon atoms, or 6-10
carbon atoms. The aryl group can have a single ring (e.g., phenyl)
or multiple condensed (fused) rings, wherein at least one ring is
aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or
anthryl). Typical aryl groups include, but are not limited to,
radicals derived from benzene, naphthalene, anthracene, biphenyl,
and the like. The aryl can be unsubstituted or optionally
substituted, as described above for alkyl groups. For example, an
aryl group can be substituted with one or more substituents (as
described above) to provide various substituted aryls, such as
pentafluorophenyl or para-trifluoromethylphenyl, and the like.
[0125] The term "halo" refers to the groups fluoro, chloro, bromo,
and iodo. Similarly, the term "halogen" refers to fluorine,
chlorine, bromine, and iodine.
[0126] The term "heteroaryl" is defined herein as a monocyclic,
bicyclic, or tricyclic ring system containing one, two, or three
aromatic rings and containing at least one nitrogen, oxygen, or
sulfur atom in an aromatic ring, and that can be unsubstituted or
substituted, for example, with one or more, and in particular one
to three, substituents, as described above in the definition of
"substituted". Typical heteroaryl groups contain 2-14 carbon atoms
in addition to the one or more heteroatoms. Examples of heteroaryl
groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl,
4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl,
.beta.-carbolinyl, carbazolyl, chromenyl, cinnolinyl,
dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl,
indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl,
isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl,
pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl,
phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl,
phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl,
pyridazinyl, pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl,
quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl,
thiazolyl, thienyl, triazolyl, tetrazolyl, and xanthenyl. In one
embodiment the term "heteroaryl" denotes a monocyclic aromatic ring
containing five or six ring atoms containing carbon and 1, 2, 3, or
4 heteroatoms independently selected from non-peroxide oxygen,
sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, aryl, or
(C.sub.1-C.sub.6)alkylaryl. In another embodiment heteroaryl
denotes an ortho-fused bicyclic heterocycle of about eight to ten
ring atoms derived therefrom, particularly a benz-derivative or one
derived by fusing a propylene, trimethylene, or tetramethylene
diradical thereto.
[0127] The term "heterocycle" refers to a saturated or partially
unsaturated ring system, containing at least one heteroatom
selected from the group oxygen, nitrogen, and sulfur, with a ring
size of 3 to about 12 atoms, or bicyclic ring systems that include
a total of about 7 to about 14 ring atoms, and optionally
substituted with one or more groups as defined herein under the
term "substituted". A heterocycle can be a monocyclic, bicyclic, or
tricyclic group containing one or more heteroatoms. A heterocycle
group also can contain an oxo group (.dbd.O) or a thioxo (.dbd.S)
group attached to the ring. Non-limiting examples of heterocycle
groups include 1,3-dihydrobenzofuran, 1,3-dioxolane, 1,4-dioxane,
1,4-dithiane, 2H-pyran, 2-pyrazoline, 4H-pyran, chromanyl,
imidazolidinyl, imidazolinyl, indolinyl, isochromanyl,
isoindolinyl, morpholine, piperazinyl, piperidine, piperidyl,
pyrazolidine, pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrroline,
quinuclidine, and thiomorpholine.
[0128] The term "heterocycle" can include, by way of example and
not limitation, a monoradical of the heterocycles described in
Paquette, Leo A.; Principles of Modern Heterocyclic Chemistry (W.
A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7,
and 9; The Chemistry of Heterocyclic Compounds, A Series of
Monographs" (John Wiley & Sons, New York, 1950 to present), in
particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.
1960, 82, 5566. In one embodiment, "heterocycle" includes a
"carbocycle" as defined herein, wherein one or more (e.g. 1, 2, 3,
or 4) carbon atoms have been replaced with a heteroatom (e.g. O, N,
or S).
[0129] Heterocycles, by way of example and not limitation, include
dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl,
tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl,
pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, piperidinyl,
4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl,
tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,
decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl,
6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl,
thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,
phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,
pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly,
purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl,
quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl,
.beta.-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,
phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl,
imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl,
isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl,
benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,
isatinoyl, and bis-tetrahydrofuranyl.
[0130] By way of example and not limitation, carbon bonded
heterocycles are bonded at position 2, 3, 4, 5, or 6 of a pyridine,
position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a
pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4,
or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or
tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or
thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or
isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4
of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or
position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Carbon bonded
heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl,
6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl,
6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl,
6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl,
2-thiazolyl, 4-thiazolyl, 5-thiazolyl, and the like.
[0131] By way of example and not limitation, nitrogen bonded
heterocycles can be bonded at position 1 of an aziridine,
azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline,
imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole,
pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine,
indole, indoline, 1H-indazole, position 2 of a isoindole, or
isoindoline, position 4 of a morpholine, and position 9 of a
carbazole, or .beta.-carboline. In one embodiment, nitrogen bonded
heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,
1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
[0132] The term "amino" refers to --NH.sub.2. The amino group can
be optionally substituted as defined herein for the term
"substituted", e.g., the amino group can be --NR.sub.2 where R is a
group recited in the definition of substituted. For example, the
groups --NR.sub.2 can include "alkylamino" wherein at least one R
is alkyl and the second R is alkyl or hydrogen, and/or "acylamino"
(--N(R)C(.dbd.O)R), wherein each R is independently hydrogen,
alkyl, alkaryl or aryl.
[0133] The term "alkaryl" refers to an aryl group substituted with
at least one alkyl group, which together form a substituent through
a radical on either the alkyl or the aryl group. The alkyl group of
the alkaryl can include about 1-8 carbon atoms, either linear or
branched. Typical alkaryl groups include benzyl, 2-phenylethyl,
3-phenylpropyl, 4-phenylbuty, 5-phenylpentyl, 6-phenylhexyl,
7-phenylheptyl, 8-phenyloctyl, branched alkyl chain derivatives
thereof, and naphthalene versions thereof. The alkaryl can be
optionally substituted as described above for alkyl groups.
[0134] The term "interrupted" indicates that another group is
inserted between two adjacent carbon atoms (and the hydrogen atoms
to which they are attached (e.g., methyl (CH.sub.3), methylene
(CH.sub.2) or methine (CH))) of a particular carbon chain being
referred to in the expression using the term "interrupted",
provided that each of the indicated atoms' normal valency is not
exceeded and the interruption results in a stable compound.
Suitable groups that can interrupt a carbon chain include, e.g.,
with one or more non-peroxide oxy (--O--), thio (--S--), imino
(--N(H)--), methylene dioxy (--OCH.sub.2O--), carbonyl
(--C(.dbd.O)--), carboxy (--C(.dbd.O)O--), carbonyldioxy
(--OC(.dbd.O)O--), carboxylato (--OC(.dbd.O)--), imine (C.dbd.NH),
sulfinyl (SO) and sulfonyl (SO.sub.2). Alkyl groups can be
interrupted by one or more (e.g., 1, 2, 3, 4, 5, or about 6) of the
aforementioned suitable groups. The site of interruption can also
be between a carbon atom of an alkyl group and a carbon atom to
which the alkyl group is attached.
[0135] As to any of the above groups, which contain one or more
substituents, it is understood, of course, that such groups do not
contain any substitution or substitution patterns that are
sterically impractical and/or synthetically non-feasible. In
addition, the compounds of this invention include all
stereochemical isomers arising from the substitution of these
compounds. It will be appreciated that some compounds of the
present invention may contain asymmetrically substituted carbon
atoms, and may be isolated in optically active or racemic forms. It
is well known in the art how to prepare optically active forms,
such as by resolution of racemic forms or by synthesis from
optically active starting materials. All chiral, diastereomeric,
racemic forms and all geometric isomeric forms of a structure are
part of this invention. In addition, some compounds of the present
invention may exist as rotational isomers. In some instances, these
rotational isomers may be separated. Both rotational isomers and a
mixture thereof are contemplated by the present invention.
[0136] One isomeric form may display superior properties or
activity compared with another. When required, separation of the
racemic material can be achieved by HPLC using a chiral column or
by a resolution using a resolving agent such as camphonic chloride
as described by Tucker et al., J. Med. Chem., 37: 2437 (1994). A
chiral compound may also be directly synthesized using a chiral
catalyst or a chiral ligand, e.g. Huffman et al., J. Org. Chem.,
60: 1590 (1995).
[0137] An "effective amount" generally means an amount that
provides a desired effect, for example, an amount sufficient to
bring about a reaction.
[0138] As used herein, "contacting" refers to the act of touching,
making contact, or of bringing to immediate or close proximity,
including at the molecular level, for example, to bring about a
chemical reaction or physical change, e.g., in a solution, cell, or
other reaction mixture.
[0139] The term "reactive group" refers to an activated ester of a
carboxylic acid, an amine, an alcohol, a sulfonyl halide, a
mercaptan, a boronate, a phosphoramidite, an isocyanate, a
haloacetamide, an aldehyde, an azide, an acyl nitrile, a
photoactivateable group or an alkyl halide.
[0140] The term "conjugated substance" refers to a covalently bound
substance such as a surface (e.g. a bead, solid support, resin
particle, or an assay plate), biological molecule (e.g., proteins,
nucleotides, polynucleotides including DNA and RNA, enzyme
substrates, antibodies, nanobodies, polypeptides, polypeptide-based
toxins, amino acids, lipids, carbohydrates, haptens, small
molecules, drugs, drug compounds, ion-complexing agents, such as
metal chelators, microparticles, synthetic or natural polymers,
cells, viruses, other fluorescent molecules or surfaces), or other
moieties of interest, e.g. a chloroalkane or a
cyanobenzothiazole.
[0141] A "tracer" is a type of conjugated substance where a dye of
the present invention is conjugated to a biological molecule as
defined above, possibly through a linker.
[0142] The term "traceless linker" or "self-immolative linker"
refers to a linker wherein cleavage of a conjugated substance from
the linker results in spontaneous cleavage of the linker from the
dye to release the unbound dye. Exemplary traceless linkers
include:
##STR00009##
[0143] As would be recognized by one of ordinary skill in the art,
further variations in linker length and substitution are
possible.
Dyes
[0144] The invention provides compounds of Formula (Ia) and
(Ib):
##STR00010##
wherein
[0145] R.sup.1 and R.sup.11 are independently H or C.sub.1-4 alkyl,
L-R or L-C.sub.S;
[0146] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0147] R is a reactive group;
[0148] C.sub.S is a conjugated substance;
[0149] R.sup.2, R.sup.5, R.sup.12 and R.sup.15 are independently H,
alkyl, aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H,
L-SO.sub.3H, L-R or L-C.sub.S;
[0150] R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25,
and R.sup.26 are independently H or C.sub.1-6 alkyl or one or more
of R.sup.20 and R.sup.21, R.sup.21 and R.sup.22, R.sup.22 and
R.sup.23, R.sup.24 and R.sup.25, R.sup.25 and R.sup.26, and
R.sup.26 and R.sup.23 together form an aryl, heteroaryl,
carbocyclic or heterocyclic ring;
[0151] R.sup.1 and R.sup.2 and/or R.sup.11 and R.sup.12 may
together form a carbocyclic, heterocyclic, aryl or heteroaryl
ring;
[0152] R.sup.6-10 are independently H, halo, OH, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0153] each X is independently CHR.sup.23, O, S or NR.sup.30;
and
[0154] R.sup.30 is H, C.sub.1-4 alkyl or --C(O)C.sub.1-4 alkyl.
[0155] In some embodiments, the ring formed by R.sup.1 and R.sup.2
or R.sup.11 and R.sup.12 can be from 3-10 atoms chosen from C, N, O
and S. In other embodiments, the ring has from 5 to 7 atoms. In
certain embodiments, the ring atoms are all carbon. These rings may
contain elements of unsaturation as well. In certain embodiments,
these rings are aryl or heteroaryl rings.
[0156] In Formula (Ib), R.sup.2 and R.sup.5 may together form an
aryl or heteroaryl ring. Suitably, the ring is phenyl or
thiophenyl. In some embodiments, the ring is substituted.
[0157] Suitably, X is CH.sub.2. R.sup.11 is suitably C.sub.1-4
alkyl. In some embodiments, R.sup.11 is methyl or ethyl. In other
embodiments, R.sup.11 and R.sup.12 form a 5-7 membered carbocyclic
ring. Suitably, the ring is an unsubstituted 6-membered ring.
Suitably, R.sup.2 and R.sup.12 are H, Cl or OMe. R.sup.5 and
R.sup.15 may be H.
[0158] The invention also provides compounds of formula (IIa) and
(IIb):
##STR00011##
wherein
[0159] R.sup.1 and R.sup.11 are independently H or C.sub.1-4 alkyl,
L-R or L-C.sub.S;
[0160] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0161] R is a reactive group;
[0162] C.sub.S is a conjugated substance;
[0163] R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.12, R.sup.13,
R.sup.14 and R.sup.15 are independently H, alkyl, aryl, heteroaryl,
CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0164] R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25
and R.sup.26 are independently H or C.sub.1-6 alkyl or one or more
of R.sup.20 and R.sup.21, R.sup.21 and R.sup.22, R.sup.22 and
R.sup.23, R.sup.24 and R.sup.25, R.sup.25 and R.sup.26, and
R.sup.26 and R.sup.23 together form an aryl, heteroaryl,
carbocyclic or heterocyclic ring;
[0165] R.sup.1 and R.sup.2 and/or R.sup.11 and R.sup.12 may
together form a carbocyclic, heterocyclic, aryl or heteroaryl
ring;
[0166] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S;
[0167] each X is independently CHR.sup.23, O, S or NR.sup.30;
and
[0168] R.sup.30 is H, C.sub.1-4 alkyl or --C(O)C.sub.1-4 alkyl.
[0169] In some embodiments, the ring formed by R.sup.1 and R.sup.2
or R.sup.11 and R.sup.12 can be from 3-10 atoms chosen from C, N, O
and S. In other embodiments, the ring has from 5 to 7 atoms. In
certain embodiments, the ring atoms are all carbon. These rings may
contain elements of unsaturation as well. In certain embodiments,
the rings may be aryl or heteroaryl rings.
[0170] In Formula (IIa), one or more of R.sup.2 and R.sup.3,
R.sup.4 and R.sup.5, R.sup.12 and R.sup.13 or R.sup.14 and R.sup.15
can also be joined together with an aryl or heteroaryl ring. In
Formula (IIb), one or both of R.sup.4 and R.sup.5 or R.sup.14 and
R.sup.15 may together form an aryl or heteroaryl ring. Suitably,
the ring is phenyl or thiophenyl. In some embodiments, the ring is
substituted.
[0171] Suitably, X is CH.sub.2. R.sup.11 is suitably C.sub.1-4
alkyl. In some embodiments, R.sup.11 is methyl or ethyl. In other
embodiments, R.sup.11 and R.sup.12 together form a 5-7 membered
carbocyclic ring. Suitably, the ring is an unsubstituted 6-membered
ring. Suitably, R.sup.2, R.sup.5, R.sup.12 and R.sup.15 are H.
[0172] The invention also provides compounds of formula (IIIa),
(IIIb) and (IIIc):
##STR00012##
wherein
[0173] R.sup.11 is independently H or C.sub.1-4 alkyl, L-R or
L-C.sub.S;
[0174] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0175] R is a reactive group;
[0176] C.sub.S is a conjugated substance;
[0177] R.sup.2 and R.sup.16 can be independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0178] R.sup.3 and R.sup.4 are H, alkyl, L-R, L-C.sub.S,
L-CO.sub.2H, L-SO.sub.3H or together form a carbocyclic, aryl,
heteroaryl, or heterocyclic ring;
[0179] alternatively, R.sup.2 and R.sup.3 and independently R.sup.4
and R.sup.16 together form a carbocyclic, heterocyclic, aryl or
heteroaryl ring;
[0180] R.sup.5, R.sup.12, R.sup.13, R.sup.14 and R.sup.15 are
independently H, alkyl, aryl, heteroaryl, CO.sub.2H, SO.sub.3H,
L-CO.sub.2H, L-SO.sub.3H, L-R or L-C.sub.S;
[0181] R.sup.20, R.sup.21, R.sup.22 and R.sup.23 are independently
H or C.sub.1-6 alkyl or one or more of R.sup.20 and R.sup.21,
R.sup.21 and R.sup.22, R.sup.22 and R.sup.23, together form an
aryl, heteroaryl, carbocyclic or heterocyclic ring;
[0182] R.sup.11 and R.sup.12 may together form a carbocyclic,
heterocyclic, aryl or heteroaryl ring;
[0183] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S;
[0184] X is CHR.sup.23, O, S or NR.sup.30; and
[0185] R.sup.30 is H, C.sub.1-4 alkyl or --C(O)C.sub.1-4 alkyl.
[0186] In formulas (Ma) and (Mb), R.sup.16 and R.sup.5 may together
form a carbocyclic, heterocyclic, aryl or heteroaryl ring. In
formula (IIIc), R.sup.14 and R.sup.5 and/or R.sup.2 and R.sup.13
may together form a carbocyclic, heterocyclic, aryl or heteroaryl
ring. Suitably, the ring is a phenyl or thiophenyl. In some
embodiments, the ring is substituted.
[0187] In some embodiments, the ring formed by R.sup.11 and
R.sup.12 can be from 3-10 atoms chosen from C, N, O and S. In other
embodiments, the ring has from 5 to 7 atoms. In certain
embodiments, the ring atoms are all carbon. These rings may contain
elements of unsaturation as well. In certain embodiments, the ring
is aryl or heteroaryl.
[0188] The invention also provides compounds according to Formula
(IV):
##STR00013##
wherein
[0189] R.sup.2 and R.sup.16 can be independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0190] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0191] R is a reactive group;
[0192] C.sub.S is a conjugated substance;
[0193] R.sup.3 and R.sup.4 are H, alkyl or together form a
carbocyclic, aryl, heteroaryl, or heterocyclic ring;
[0194] alternatively, R.sup.2 and R.sup.3 and independently R.sup.4
and R.sup.16 together form a carbocyclic, heterocyclic, aryl or
heteroaryl ring;
[0195] R.sup.5 and R.sup.15 are independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0196] R.sup.22 and R.sup.23 are independently H or C.sub.1-6 alkyl
or together form an aryl, heteroaryl, carbocyclic or heterocyclic
ring; and
[0197] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S.
[0198] The invention further provides compounds according to
Formula (V):
##STR00014##
wherein
[0199] R.sup.2 and R.sup.16 can be independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0200] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0201] R is a reactive group;
[0202] C.sub.S is a conjugated substance;
[0203] R.sup.3 and R.sup.4 are H, alkyl, or together form a
carbocyclic, heterocyclic, aryl or heteroaryl ring;
[0204] alternatively, R.sup.2 and R.sup.3 and independently R.sup.4
and R.sup.16 may together form a carbocyclic, heterocylic, aryl or
heteroaryl ring;
[0205] R.sup.5 and R.sup.15 are independently H, alkyl, aryl,
heteoaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0206] R.sup.20 and R.sup.21 are independently H or C.sub.1-6 alkyl
or together form an aryl, heteroaryl, carbocyclic or heterocyclic
ring; and
[0207] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S.
[0208] The invention additionally provides compounds according to
Formula (VI):
##STR00015##
wherein
[0209] R.sup.5 and R.sup.15 are independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0210] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0211] R is a reactive group;
[0212] C.sub.S is a conjugated substance;
[0213] R.sup.22, R.sup.23, R.sup.26 and R.sup.27 are independently
H or C.sub.1-6 alkyl or one or more of R.sup.22 and R.sup.23 and
R.sup.26 and R.sup.27 together form an aryl, heteroaryl,
carbocyclic or heterocyclic ring; and
[0214] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S.
[0215] The invention additionally provides compounds according to
Formula (VII):
##STR00016##
wherein
[0216] R.sup.5 and R.sup.15 are independently H, alkyl, aryl,
heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0217] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0218] R is a reactive group;
[0219] C.sub.S is a conjugated substance;
[0220] R.sup.20, R.sup.21, R.sup.24 and R.sup.25 are independently
H or C.sub.1-6 alkyl or one or more of R.sup.20 and R.sup.21 and
R.sup.24 and R.sup.25 together form an aryl, heteroaryl,
carbocyclic or heterocyclic ring; and
[0221] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, heteroaryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H,
L-R or L-C.sub.S.
[0222] The following statements apply to Formulae (I)-(VII), where
appropriate. Suitably, R.sup.2, R.sup.5 and R.sup.15 are H. R.sup.3
and R.sup.4 are suitably C.sub.1-4 alkyl. In some embodiments,
R.sup.3 and R.sup.4 are methyl or ethyl. R.sup.3 and R.sup.4 may
together form a heterocycle, such as a piperazine. In other
embodiments, R.sup.2 and R.sup.3 and/or R.sup.4 and R.sup.16
together form a 5-7 membered carbocylic ring. The ring is suitably
an unsubstituted 6-membered ring.
[0223] R.sup.10 is suitably H, F, Cl, CO.sub.2H or SO.sub.3H. In
certain embodiments, R.sup.10 is H. R.sup.6 and R.sup.9 are
suitably H or halo. In certain embodiments, R.sup.6 and R.sup.9 may
be either Cl or F.
[0224] Suitably, one of R.sup.7 and R.sup.8 is -L-R, -L-CO.sub.2H
or -L-C.sub.S and the other is H, Cl, or F.
[0225] L is suitably --CO--, --SCH.sub.2CO-- or --SO.sub.2--. L may
also contain a PEG moiety. In other embodiments, L is a "traceless"
or "self-immolative" linker.
[0226] Suitably, R is
##STR00017##
with SE most preferred.
[0227] C.sub.s is suitably a chloroalkane of the formula
NHCH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.n(CH.sub.2).sub.6Cl, with
n being 2-6; a nucleoside, for example
##STR00018##
an oligonucleotide suitably attached through allylaminodU; or a
cyanobenzothiazole. Suitably, the cyanobenzothiazole is
##STR00019##
wherein Z is O or NH.
[0228] Suitable compounds include those shown in FIGS. 1-3. As one
of ordinary skill in the art would understand, any of the
combinations between column 1 and row 1 are feasible. Those that
show a compound number have been synthesized.
[0229] Among other unique properties, compounds of the present
invention containing a 7-membered ring show a significant red shift
in the excitation and emission of the dye as compared to prior art
compounds. In certain embodiments, the compounds of the present
invention have emission maxima from about 600 nm to about 730 nm.
In certain embodiments, the compounds of the present invention have
excitation maxima from about 575 nm to about 675 nm.
[0230] Although there is no description of incorporating a fused
7-membered ring in the manner described here into a fluorescent dye
in the literature, two publications (Zachariasse, et al., J.
Photochem. Photobiol. A, 1997, 105, 373-383; Saha and Samanta, J.
Phys. Chem. A 2002, 106, 4763-4771) describe comparisons between
incorporating a nitrogen involved in dye fluorescence in a
six-membered carbocyclic ring and a seven-membered carbocyclic
ring. With the dyes described in these literature reports, as the
ring size is increased, the quantum yield drops dramatically with
increasing solvent polarity, which does not occur for the dyes of
the present invention.
[0231] There has also been a discussion of the effect of unfused
ring size in rosamine derivatives (Lu and Burgess, J. Org. Chem.,
2008, 73, 8711-8718). No shift in emission wavelength was observed
on varying the ring size from a five- to a six-membered ring. The
red-shifted emission of the dyes containing a fused seven-membered
ring relative to those with six-membered rings while maintaining a
high quantum yield in polar solvents was unanticipated.
Synthesis of Dyes
[0232] Compounds described herein may be synthesized and conjugated
using a variety of methods. See, e.g., Beija, et al., Chem. Soc.
Rev., 2009, 38, 2410-2433. Exemplary syntheses are generalized
below.
Synthesis of Dye Precursors:
##STR00020##
[0234] Synthesis of intermediate 3 can be done through formation of
an oxime of tetralone 1 followed by a Beckmann rearrangement to
give compound 2. Lactam reduction then provides compound 3.
##STR00021##
[0235] Alkylation of this cyclic amine 3 followed by demethylation
provides compound 4.
##STR00022##
[0236] Alkylation of the nitrogen with bromochloropropane followed
by heat induced cyclization provides a tricyclic amine.
Demethylation of this amine provides compound 5. Use of the
5-methoxy tetralone provides the isomeric aminophenol.
Synthesis of Symmetrical Dyes:
##STR00023##
[0238] Synthesis of symmetrical rhodamines is accomplished through
a melt procedure where compound 3, anhydride 6 and ZnCl.sub.2 are
fused together with high heat. When anhydride 6 is unsymmetrical,
purification provides both isomeric dyes, typically in equal
amounts. Often, some of the decarboxylated rosamine is also
isolated resulting from heat induced decarboxylation of compound
1a.
Synthesis of Unsymmetrical Dyes:
##STR00024##
[0240] Synthesis of unsymmetrical rhodamine dyes is accomplished in
a two step procedure. In the first step, an aminophenol 3 is
reacted with an anhydride 6 in the absence of an acidic catalyst to
provide the ketone adduct 7. As above, if 6 is unsymmetrical the
two isomers of 7 can be separated at this stage.
##STR00025##
[0241] Compound 7 is then reacted with another aminophenol (or
aminonaphthol) to give a dye of formula Ma. Typical procedure for
this reaction is in DMF at 80.degree. C. with catalytic
trimethylsilylpolyphosphate.
Conjugation of Halogenated Dyes:
##STR00026##
[0243] Tetrahalogenated rhodamines (either fluoro or chloro) were
functionalized by treatment with mercaptoacetic acid in DMF. This
carboxylic acid could then be activated as an succidimidyl ester
(SE) and linked to biomolecules. Rhodamines with a carboxylic acid
on the lower ring were directly activated as an SE and linked to
biomolecules.
[0244] As can be appreciated by the skilled artisan, alternative
methods of synthesizing the compounds of the formulae herein will
be evident to those of ordinary skill in the art. Additionally, the
various synthetic steps may be performed in an alternate sequence
or order to give the desired compounds. Synthetic chemistry
transformations and protecting group methodologies (protection and
deprotection) useful in synthesizing the compounds described herein
are known in the art and include, for example, those such as
described in R. Larock, Comprehensive Organic Transformations, VCH
Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective
Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991);
L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic
Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,
Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons
(1995), and subsequent editions thereof.
Intermediates
[0245] The invention further provides compounds of formulae (VIII)
and (IX):
##STR00027##
wherein
[0246] R.sup.11 is H or C.sub.1-4 alkyl, L-R or L-C.sub.S;
[0247] L is a covalent linkage that is linear or branched, cyclic
or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester,
acid, amine, amide, alcohol, ether, thioether or halide groups or
single, double, triple or aromatic carbon-carbon bond;
[0248] R is a reactive group;
[0249] C.sub.S is a conjugated substance;
[0250] R.sup.12 and R.sup.15 are independently H, alkyl, aryl,
CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0251] R.sup.20, R.sup.21, R.sup.22 and R.sup.23 are independently
H or C.sub.1-6 alkyl or one or more of R.sup.20 and R.sup.21,
R.sup.21 and R.sup.22 and R.sup.22 and R.sup.23 together form a
fused aryl ring;
[0252] R.sup.11 and R.sup.12 may be joined together in an
optionally substituted ring;
[0253] R.sup.6-10 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, L-SO.sub.3H, L-R or
L-C.sub.S;
[0254] X is CHR.sup.23, O, S or NR.sup.30; and
[0255] R.sup.30 is H, C.sub.1-4 alkyl or --C(O)C.sub.1-4 alkyl.
[0256] In some embodiments, the ring formed by R.sup.11 and
R.sup.12 can be from 3-10 atoms chosen from C, N, O and S. These
rings may contain elements of unsaturation as well.
[0257] These compounds are useful in the synthesis of compounds of
formulae (I)-(VII).
Labeled Biomolecules
[0258] Briefly, the dyes of the present invention may be used as
labeling agents which allow for the detection of a composition of
matter. The dyes of the present invention can be used to label a
broad range of molecules, including but not limited to,
biomolecules such as polypeptides, polypeptide-based toxins, amino
acids, nucleotides, polynucleotides including DNA and RNA, lipids,
carbohydrate, and enzyme substrates. Additionally, the compounds
may be used to label haptens, small molecules, drugs, drug
compounds, ion-complexing agents, such as metal chelators,
microparticles, synthetic or natural polymers, cells, viruses,
other fluorescent molecules or surfaces. The resulting labeled
molecules may be referred to as conjugates or tracers.
[0259] In some aspects, the dyes can be conjugated with a
nucleoside, nucleotide or a polynucleotide. The dyes of the
invention may be conjugated with nucleoside, nucleotide or
polynucleotide in any way known to one of ordinary skill in the art
such as through a phosphoramidite, an activated ester or a reactive
platinum complex.
[0260] In other aspects, the dyes of the invention can be used to
conjugate with an amino acid, amino acid analog, or polypeptide. In
other aspects, the dyes of the invention can be used to conjugate
with a small molecule, e.g., a drug or drug compound. In some
aspects, the conjugated small molecule can be used as a fluorescent
tracer.
[0261] In some embodiments, the labeled biomolecules may be
profluorescent compounds. A profluorescent compound is one that has
a fluorescence that is reduced as compared to the related dye and
contains a substrate for an enzyme of interest. Upon being acted on
by the enzyme of interest, the fluorescent dye from the
profluorescent compound is released, and thereby fluorescence is
generated. A "traceless" or "self-immolative" linker can also be
included between the dye and the enzyme substrate.
[0262] Exemplary labeled biomolecules include compounds of formulae
(XIa), (XIb) and (XIc):
##STR00028##
wherein
[0263] R.sup.11 is H or C.sub.1-6 alkyl;
[0264] W is a traceless linker or a direct bond;
[0265] R is a reactive group;
[0266] C.sub.S is an enzyme substrate;
[0267] R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.11, R12, R.sup.13,
R.sup.14, R.sup.15 and R.sup.16 are independently H, alkyl, aryl,
CO.sub.2H, SO.sub.3H, L-R, L-C.sub.S, L-CO.sub.2H, or
L-SO.sub.3H;
[0268] R.sup.11 and R.sup.12 may be joined together in an aryl,
heteroaryl, carbocyclic or heterocyclic ring ring;
[0269] R.sup.6-9 are independently H, F, Cl, Br, I, OH, alkyl,
aryl, CO.sub.2H, SO.sub.3H, L-CO.sub.2H, or L-SO.sub.3H; and
[0270] X is CH.sub.2, 0, 5 or NH.
[0271] In some embodiments, the enzyme substrate is Z-DEVD. In
another embodiment, the enzyme substrate is Z-AAF-benzyl.
[0272] Profluorescent compounds according to formulae (XIa)
include:
##STR00029##
[0273] In other embodiments, the invention provides other
profluorescent biomolecules. In some embodiments, a dye according
to the present invention is attached through a biomolecule to a
quencher. Any quencher that absorbs in the emission spectrum of the
dye can be used. One of ordinary skill in the art would be able to
identify such quenchers. Suitable quenchers include Black Hole
Quenchers.TM. and QXL.TM. quenchers. (Available from Life
Technologies).
[0274] Exemplary profluorescent compounds containing a quencher
include: Quencher-GABA-Pro-Cha-Abu-Smc-His-Ala-Dab(dye)-Ala-Lys-NH2
as an MMP-3 substrate where cleavage in the amino acid chain
separates the quencher from the dye, producing fluorescence. In one
embodiment, the dye can be conjugated to the amino acid chain
through a carboxylic acid in the lower ring. The dye can also be
attached to the amino acid chain at other positions.
Methods of Use
[0275] The dyes of the present invention provide an effective tool
for covalently labeling biomolecules for a wide variety of
applications. Labeling allows one to study interactions involving
biomolecules such as proteins, glycoproteins, nucleic acids and
lipids, as well as small molecules, e.g., drugs or drug compounds,
inorganic chemicals or any combinations thereof. The interactions
may be studied in cell-free biological systems, in cellular systems
or in vivo. Analyzing the various interactions is often a
significant part of scientific research and development, drug
design, screening and optimization, phylogenetic classification,
genotyping individuals, parental and forensic identification,
environmental studies, diagnosis, prognosis, and/or treatment of
disease conditions.
[0276] In some aspects of the invention, the conjugates of the
invention are used to label a sample so that the sample can be
identified or quantitated. For instance, such conjugates may be
added as part of an assay for a biological target analyte or as a
detectable tracer element in a biological or non-biological
fluid.
[0277] The sample may be obtained directly from biological
materials, e.g., a wash from a solid material (organic or
inorganic), a medium in which cells have been cultured, a cell
lysate, a buffer solution in which cells have been placed for
evaluation, or physiological sources, e.g., blood, plasma, serum,
urine, etc. When the sample comprises cells, the cells are
optionally single cells, including microorganisms, or multiple
cells associated with other cells in two or three dimensions,
including multicellular organisms, embryos, tissues, biopsies,
filaments, biofilms, and the like. When the sample comprises cells,
the cells may be lysed, e.g., a cell lysate, or whole cells.
[0278] Alternatively, the sample is a solid, optionally a smear or
scrape or a retentate removed from a liquid or vapor by filtration.
In one aspect of the invention, the sample is obtained from a
biological fluid, including separated or unfiltered physiological
fluids such as urine, cerebrospinal fluid, blood, lymph fluids,
tissue homogenate, interstitial fluid, cell extracts, mucus,
saliva, sputum, stool, physiological secretions or other similar
fluids. Alternatively, the sample is obtained from an environmental
source such as soil, water, or air; or from an industrial source
such as taken from a waste stream, a water source, a supply line,
or a production lot.
[0279] In other embodiments, the sample is present on or in a solid
or semi-solid matrix. In one aspect of the invention, the matrix is
a membrane. In other aspects, the matrix is an electrophoretic gel,
such as those used for separating and characterizing nucleic acids
or proteins, or a blot prepared by transfer from an electrophoretic
gel to a membrane. In other aspects, the matrix is a silicon chip
or glass slide, and the analyte of interest has been immobilized on
the chip or slide in an array (e.g., the sample comprises proteins
or nucleic acid polymers in a microarray). In yet other aspects,
the matrix is a microwell plate or microfluidic chip, and the
sample is analyzed by automated methods, typically by various
methods of high-throughput screening, such as drug screening.
[0280] The dye conjugates are generally utilized by combining the
conjugate as described above with the sample of interest under
conditions selected to yield a detectable optical response. The
sample is then illuminated at a wavelength selected to elicit the
optical response. Typically, a specified characteristic of the
sample is determined by comparing the optical response with a
standard or expected response.
[0281] A detectable optical response means a change in, or
occurrence of, an optical signal that is detectable either by
observation or instrumentally. Typically, the detectable response
is a change in fluorescence, such as a change in the intensity,
excitation or emission wavelength distribution of fluorescence,
fluorescence lifetime, fluorescence polarization, or a combination
thereof. The degree and/or location of the labeling, compared with
a standard or expected response, indicates whether, and to what
degree, the sample possesses a given characteristic. Some dyes of
the invention may exhibit little fluorescence emission, but are
still useful as chromophoric dyes. Such chromophores are useful as
energy acceptors in FRET applications, or to simply impart the
desired color to a sample or portion of a sample.
[0282] For biological applications, the dye conjugates are
typically used in an aqueous, mostly aqueous or aqueous-miscible
solution prepared according to methods generally known in the art.
The exact concentration of the dye compound is dependent upon the
experimental conditions and the desired results, but typically
ranges from about one nanomolar to one millimolar or more. The
optimal concentration may be determined by systematic variation
until satisfactory results, with minimal background fluorescence,
are accomplished.
[0283] The dye conjugates may be used to label samples containing
biological components. The sample may comprise heterogeneous
mixtures of components (including intact cells, cell extracts, cell
lysates, bacteria, viruses, organelles, and mixtures thereof) or a
single component or homogeneous group of components (e.g., natural
or synthetic amino acid, nucleic acid or carbohydrate polymers, or
lipid membrane complexes). The dyes are generally non-toxic to
living cells and other biological components within the
concentrations of use.
[0284] The dye conjugate may be combined with the sample in a way
that facilitates contact between the dye conjugate and the sample
components of interest. Typically, the dye conjugate or a solution
containing the dye conjugate is simply added to the sample. Certain
dyes of the invention, e.g., those that are substituted by one or
more sulfonic acid moieties, may be less permeant to membranes of
biological cells, but once inside viable cells are typically well
retained. Treatments that permeabilize the plasma membrane, such as
electroporation, shock treatments or high extracellular ATP, may be
used to introduce selected dye conjugates into cells.
Alternatively, selected dye conjugates can be physically inserted
into cells, e.g., by pressure microinjection, scrape loading, patch
clamp methods, or phagocytosis.
[0285] Dyes that incorporate an aliphatic amine or a hydrazine
residue may be microinjected into cells where they can be fixed in
place by aldehyde fixatives such as formaldehyde or glutaraldehyde.
This property makes such dyes useful for intracellular applications
such as neuronal tracing.
[0286] Dyes that possess a lipophilic substituent, such as
phospholipids, may non-covalently incorporate into lipid
assemblies, e.g., for use as probes for membrane structure, or for
incorporation in liposomes, lipoproteins, films, plastics,
lipophilic microspheres or similar materials; or for tracing.
Lipophilic dyes are useful as fluorescent probes of membrane
structure.
[0287] Chemically reactive dye compounds may covalently attach to a
corresponding functional group on a wide variety of materials to
form dye conjugates as described above. Using dye compounds to
label reactive sites on the surface of cells, in cell membranes, in
intracellular compartments such as organelles, or in the cytoplasm,
permits the determination of their presence or quantity,
accessibility, activity or their spatial and temporal distribution
in the sample. Photoreactive dyes may be used similarly to
photolabel components of the outer membrane of biological cells or
as photo-fixable polar tracers for cells.
[0288] In some embodiments, chloroalkane-labeled dyes may be used
with HaloTag 0 protein to detect proteins of interest by generating
a fusion protein between the HaloTag.RTM. protein and the protein
of interest. Generally, these fusion proteins are expressed by a
cell from a HaloTag.RTM. fusion construct, and the fusion protein
is detected through use of the chloroalkane-labeled dye. This
allows detection of protein expression or determination of a
protein expression time-course, protein localization or migration.
In addition, these proteins can be detected in gels using this
fluorescent label. The dyes may also be utilized in other
orthogonal labeling systems, such as cutinase, dihydrofolate
reductase/trimethoprim SNAP-tag, Clip Tag, Alkyl cytosine
transferase (see U.S. Patent Application No. 2012/0237961, which is
incorporated by reference herein) and Acyl Carrier Protein (see
U.S. Patent Application No. 2010/0173384, which is incorporated by
reference herein). In addition, HaloTag is also orthogonal to other
labeling chemistries such as Hsuingen cyclizations (click
chemistry), hydrazone and oxime formation and the Staudinger
ligation.
[0289] Optionally, the sample is washed after labeling to remove
residual, excess or unbound dye compound or dye conjugate. The
sample is optionally combined with one or more other solutions in
the course of labeling, including wash solutions, permeabilization
and/or fixation solutions, and solutions containing additional
detection reagents. An additional detection reagent typically
produces a detectable response due to the presence of a specific
cell component, intracellular substance, or cellular condition,
according to methods generally known in the art. When the
additional detection reagent has, or yields a product with,
spectral properties that differ from those of the subject dye
compounds, multi-color applications are possible. This is
particularly useful where the additional detection reagent is a dye
or dye conjugate having spectral properties that are detectably
distinct from those of the labeling dye.
[0290] The dye conjugates are used according to methods known in
the art, e.g., use of antibody conjugates in microscopy and
immunofluorescent assays; or nucleotide or oligonucleotide
conjugates for nucleic acid hybridization assays, nucleic acid
amplification reactions, and nucleic acid sequencing (e.g., U.S.
Pat. Nos. 5,332,666, 5,171,534, and 4,997,928, and WO 94/05688).
Dye conjugates of multiple independent dyes of the invention
possess utility for multi-color applications.
[0291] At any time after or during labeling, the sample is
illuminated with a wavelength of light selected to give a
detectable optical response and observed with a means for detecting
the optical response. Equipment that is useful for illuminating the
dye compounds of the invention includes, but is not limited to,
hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers
and laser diodes. These illumination sources are optionally
integrated into laser scanners, fluorescence microplate readers,
standard or minifluorometers, or chromatographic detectors.
[0292] The optical response is optionally detected by visual
inspection or by use of any of the following devices: CCD cameras,
video cameras, photographic film, laser-scanning devices,
fluorometers, photodiodes, quantum counters, epifluorescence
microscopes, scanning microscopes, flow cytometers, fluorescence
microplate readers, or by means for amplifying the signal such as
photomultiplier tubes. Where the sample is examined using a flow
cytometer, examination of the sample optionally includes sorting
portions of the sample according to their fluorescence
response.
Exemplary Methods of Use
[0293] i. Detection of Nucleic Acid Polymers
[0294] In one embodiment, a dye oligonucleotide conjugate of the
present invention is combined with a sample that contains, or is
thought to contain, a nucleic acid polymer, incubating the mixture
of dye oligonucleotide conjugate, e.g., probe or primer, and sample
for a time sufficient for the oligonucleotide in the conjugate to
combine with nucleic acid polymers in the sample to form nucleic
acid hybrids (complexes) (i.e., a probe), or to prime nucleic acid
synthesis (i.e., a primer), which may be detected. The
characteristics of the labeled molecules, including the presence,
location, intensity, excitation and emission spectra, fluorescence
polarization, fluorescence lifetime, and other physical properties
of the fluorescent signal, can be used to detect, differentiate,
sort, quantitate, sequence and/or analyze aspects or portions of
the sample. The dye conjugates of the invention are optionally used
in conjunction with one or more additional reagents (e.g.,
detectably different fluorescent reagents) including dyes of the
same class having different spectral properties.
[0295] Typically, the dye conjugate is prepared for use by
dissolving the dye conjugate in an aqueous or aqueous miscible
solution that is compatible with the sample and intended use. For
biological samples, where minimal perturbation of cell morphology
or physiology is desired, the solution is selected accordingly.
[0296] The labeling solution is made by dissolving the dye
conjugate directly in an aqueous solvent such as water, a buffer
solution, such as buffered saline (preferably non-phosphate for
some viability discrimination applications), a
Tris(hydroxymethyl)-aminomethane (TRIS) buffer (preferably
containing EDTA), or a water-miscible organic solvent such as
dimethylsulfoxide (DMSO), dimethylformamide (DMF), or a lower
alcohol such as methanol or ethanol. The dye conjugate is usually
preliminarily dissolved in an organic solvent (e.g., 100% DMSO) at
a concentration of greater than about 100 times that used in the
labeling solution, then diluted one or more times with an aqueous
solvent such as water or buffer, such that the dye conjugate is
present in an effective amount.
[0297] Typically labeling solutions for cellular samples have a dye
concentration greater than 0.1 nM and less than 50 .mu.M, more
typically greater than 1 nM and less than 10 .mu.M, e.g., between
0.5 and 5 .mu.M. Labeling solutions for electrophoretic gels
typically have a dye concentration of greater than 0.1 .mu.M and
less than 10 .mu.M, more typically about 0.5 to 2 .mu.M. The same
holds true when the dye is added to the gel before being combined
with nucleic acids. Labeling solutions for detection and
quantitation of free nucleic acids in solution typically have a
concentration of 0.1 .mu.M to 2 .mu.M. The optimal concentration
and composition of the labeling solution is determined by the
nature of the sample (including physical, biological, biochemical
and physiological properties), the nature of the dye-sample
interaction (including the transport rate of the dye to the site of
the nucleic acids), and the nature of the analysis being performed,
and can be determined according to standard procedures.
[0298] The nucleic acid in the sample may be DNA or RNA, or a
mixture or a hybrid thereof. Any DNA is optionally single-,
double-, triple-, or quadruple-stranded DNA; any RNA is optionally
single stranded ("ss") or double stranded ("ds"). The nucleic acid
may be a natural polymer (biological in origin) or a synthetic
polymer (modified or prepared artificially). The nucleic acid
polymer (for instance, one containing at least 8 bases or base
pairs) may be present as nucleic acid fragments, oligonucleotides,
or larger nucleic acid polymers with secondary or tertiary
structure. The nucleic acid is optionally present in a condensed
phase such as a chromosome. The nucleic acid polymer optionally
contains one or more modified bases or links or contains labels
that are non-covalently or covalently attached. For example, the
modified base can be a naturally occurring modified base such as
.PSI. (pseudouridine) in tRNA, 5-methylcytosine,
6-methylaminopurine, 6-dimethylaminopurine, 1-methylguanine,
2-methylamino-6-hydroxypurine, 2-dimethylamino-6-hydroxypurine,
5-amino-DU, isoC, isoG, or other known minor bases (see, e.g.,
Davidson, The Biochemistry Of The Nucleic Acids (1976)) or is
synthetically altered to contain an unusual linker such as
morpholine derivatized phosphates (AntiVirals, Inc., Corvallis,
Oreg.), or peptide nucleic acids such as N-(2-aminoethyl)glycine
units (Wittung et al., Nature, 368:561 (1994)) or contain a simple
reactive functional group (<10 carbons) that is an aliphatic
amine, carboxylic acid, alcohol, thiol or hydrazine, or contain a
fluorescent label or other hapten, such as inosine,
bromodeoxyuridine, iododeoxyuridine, biotin, digoxigenin,
2,4-dinitrophenyl, where the label is originally attached on the
nucleotide (e.g., CHROMATIDE.TM. labeled nucleotides, Molecular
Probes, Eugene, Oreg.) or located on the 3' or 5' end of a nucleic
acid polymer, or ligands non-covalently attached to the nucleic
acids. The sensitivity of the dyes for nucleic acid polymers
containing primarily modified bases and links may be diminished by
interference with the binding mode. Some embodiments of the dyes
may inhibit non-specific nuclease activity but not restriction
endonuclease activity at certain dye:base pair ratios.
[0299] The sample that contains a nucleic acid is optionally a
biological structure (i.e., an organism or a discrete unit of an
organism), or a solution (including solutions that contain
biological structures), or a solid or semi-solid material.
Consequently, the nucleic acid is optionally free in solution,
immobilized in or on a solid or semi-solid material, extracted from
a biological structure (e.g., from lysed cells, tissues, organisms
or organelles), or remains enclosed within a biological structure.
In order for the nucleic acids to bind to the dyes, it is necessary
that the nucleic acids be in an aqueous environment to allow
contact with the dye, even if the nucleic acids are not enclosed in
a biological structure.
[0300] The biological structure that contains the nucleic acid is
optionally a cell or tissue, for example, where the nucleic acid is
present in a cell or interstitial space, as a prokaryote or
eukaryote microorganism, or as a virus, viroid, chromosome or
organelle. Alternatively, the biological structure may not be
contained in a tissue or cell and is present either as a virus or
as a microorganism or other cell, or is present as a cellular
component removed from its parent cell (e.g., a plasmid or
chromosome, or a mitochondrion or nucleus or other organelle).
Typically, the biological structure is an organelle, chromosome or
cell that is optionally contained within a eukaryote cell. The cell
present inside a eukaryote cell is typically a parasite or other
infectious agent such as a virus, bacterium, protozoa, mycoplasma
or mycobacterium. When the nucleic acid is contained in a
biological structure that is a cell, the cells are viable or dead
cells or a mixture thereof, i.e., the integrity of the cell
membrane is optionally intact or disrupted by natural (autolytic),
mechanical or chemical means or by environmental means such as
changes in temperature or pressure. Alternatively, the cells are
blebbing or undergoing apoptosis or in a cycle of growth or cell
division.
[0301] When the nucleic acid is present in a solution, the sample
solution can vary to contain one of purified or synthetic nucleic
acids such as oligonucleotides to crude mixtures such as cell
extracts or homogenates or other biological fluids, or dilute
solutions from biological, industrial, or environmental sources. In
some cases, it is desirable to separate the nucleic acids from a
mixture of biomolecules or fluids in the solution prior to
combination with the dye. Numerous techniques exist for separation
and purification of nucleic acids from generally crude mixtures
with other proteins or other biological molecules. These include
such means as chromatographic techniques and electrophoretic
techniques using a variety of supports or solutions or in a flowing
stream. Alternatively, mixtures of nucleic acids may be treated
with RNase or DNase so the nucleic acid polymer is not degraded in
the presence of the nuclease can be discriminated from degradation
products using the subject dyes.
[0302] The relatively low toxicity of the dyes of the invention to
living systems generally enables the examination of nucleic acids
in living samples with little or no damage caused by the dye
itself. For use with intact cells or samples in a gel, more
permeant dyes may be employed, although some cells readily take up
dyes that have been shown to be impermeant to other cells by means
other than passive diffusion across cell-membranes, e.g., by
phagocytosis or other types of ingestion. These dyes can be used in
standard gel-based applications. The photostability, toxicity,
binding affinity, quantum yield, and fluorescence enhancement of
dyes are determined according to standard methods known in the
art.
[0303] In one embodiment, a dye oligonucleotide conjugate, e.g.,
probe or primer, is employed in methods and kits for the
identification of alleles in a physiological sample. In one
embodiment, an appropriate set of loci, primers, and amplification
protocols is selected to generate amplified alleles from multiple
co-amplified loci which, in one embodiment, do not overlap in size
or which are labeled in a way which enables one to differentiate
between the alleles from different loci which overlap in size. In
addition, this method contemplates the selection of short tandem
repeat (STR) loci which are compatible for use with a single
amplification protocol. Successful combinations can be generated by
trial and error of locus combinations, by selection of primer pair
sequences, and by adjustment of primer concentrations to identify
an equilibrium in which all included loci may be amplified. The
number of loci which may be amplified in a multiplex amplification
reaction step may be from 2 to 50, or any integer between 2 and 50,
e.g. 16, 17, 18, 21, 23, or 26, so long as the reaction produces
amplified alleles that can be identified. In one embodiment, the
amplified fragments are less than 500 by in length.
[0304] Synthesis of the primers used in the present method can be
conducted using any standard procedure for oligonucleotide
synthesis known to those skilled in the art. At least one primer
for each locus is covalently attached to a different dye label.
[0305] Samples of genomic DNA can be prepared for use in the method
of this invention using any method of DNA preparation which is
compatible with the amplification of DNA. Many such methods are
known by those skilled in the art. When the at least one DNA sample
to be analyzed is human genomic DNA, the DNA may be prepared from
samples, selected from the group consisting of tissue, blood,
semen, vaginal cells, hair, saliva, urine, bone, buccal samples,
amniotic fluid containing placental cells or fetal cells, chorionic
villus, and mixtures of any of the samples listed above.
[0306] Once a sample of genomic DNA is prepared, the targeted loci
can be co-amplified in the multiplex amplification step. Any one of
a number of different amplification methods can be used to amplify
the loci, including, but not limited to, polymerase chain reaction
(PCR), transcription based amplification and strand displacement
amplification (SDA). In one embodiment, the DNA sample is subjected
to PCR amplification using primer pairs specific to each locus in
the set.
[0307] At least one primer for each locus can be covalently
attached to a dye label, one of which comprises a dye of the
present invention. The primers and dyes attached thereto are
selected for use in the multiplex amplification reaction such that
the alleles amplified using primers for each locus labeled with one
color do not overlap with the alleles of the other loci in the set
co-amplified therein using primers labeled with the same color,
when the alleles are separated, e.g., by gel or capillary
electrophoresis. Fluorescent labels suitable for attachment to
primers for use in the present invention are commercially
available. See, e.g. fluorescein and carboxy-tetramethylrhodamine
labels and their chemical derivatives from PE Biosystems and
Molecular Probes. In one embodiment, at least three different
labels are used to label the different primers used in the
multiplex amplification reaction. When a size marker is included to
evaluate the multiplex reaction, the primers used to prepare the
size marker may be labeled with a different label from the primers
used to amplify the loci of interest in the reaction.
[0308] Once a set of amplified alleles is produced from the
multiplex amplification step, the amplified alleles are evaluated.
The evaluation step of this method can be accomplished by any one
of a number of different means. Electrophoresis may be used to
separate the products of the multiplex amplification reaction,
e.g., capillary electrophoresis or denaturing polyacrylamide gel
electrophoresis. Gel preparation and electrophoresis procedures and
conditions for suitable for use in the evaluating step are known to
the art. Separation of DNA fragments in a denaturing polyacrylamide
gel and in capillary electrophoresis occurs based primarily on
fragment size.
[0309] Once the amplified alleles are separated, the alleles and
any other DNA in the gel or capillary (e.g., DNA size markers or an
allelic ladder) can then be visualized and analyzed. In one
embodiment, the method for detection of multiplexes containing
numerous loci is fluorescence, where primers for each locus in the
multiplexing reaction is followed by detection of the labeled
products employing a fluorometric detector.
[0310] ii. Cell Imaging
[0311] Fluorescently-labeled biomolecules have proven extremely
useful as reporters for gene expression studies in both cultured
cells and entire animals. For example, in living cells,
fluorescently-labeled proteins are most commonly utilized to track
the localization and dynamics of proteins, organelles, and other
cellular compartments, as well as a tracer of intracellular protein
trafficking Quantitative imaging of labeled biomolecules according
to the present invention is readily accomplished with a variety of
techniques, including widefield, confocal, and multiphoton
microscopy and provides a unique window for exposing the
intricacies of cellular structure and function. Among other things,
the dyes of the present invention can be used to image subcellular
protein translocation, to detect protein-protein and protein-DNA
complexes, and to determine protein expression, localization and
activity state.
[0312] In one embodiment, the cell is contacted with a labeled
biomolecule according to the present invention, and fluorescence is
detected. For example, a protein can be labeled with a dye of the
present invention and used to bind to a cell surface receptor. In
this example, the location of the cell surface receptor can be
detected.
[0313] Alternatively, fluorescent dyes according to the present
invention can be used in a modular protein tagging system such as
HaloTag.RTM. protein (Promega, Madison Wis.). In this type of
system, the protein tag is a modified haloalkane dehalogenase
designed to covalently bind to a synthetic ligand which has a
chloroalkane linker attached to a fluorescent dye according to the
present invention.
[0314] iii Enzyme Assays
[0315] The dyes of the present invention can also be conjugated to
enzyme substrates and used to detect the presence and/or activity
of an enzyme in a sample. Thus, the invention provides a method of
detecting an enzyme in a sample. In one embodiment, a sample
suspected of containing an enzyme is contacted with a labeled
biomolecule which is a profluorescent form of a dye of the present
invention and a substrate for the enzyme; and fluorescence is
detected.
[0316] iv. Other Uses
[0317] The fluorescent dyes of the present invention can be used in
other techniques known to those skilled in the art. For example,
the dyes may be used in antibody staining, in studies of
organometallic catalysis in living cells, in biomedical imaging, in
in vivo detection of small molecules, thiol-reactive probes, biotin
and hapten derivatives, nucleic acid and protein analysis, for
probing cellular structure (including cytoskeletal proteins,
organelles, lipids and membranes and as fluorescent tracers of cell
morphology and fluid flow), and for probing cellular function
(including cell viability, cell proliferation, endocytosis,
receptors, ion channels, signal transduction, ROS, various cations,
and membrane potential). The dyes may also be used to detect
biological phenomena using FRET or BRET. See, e.g. "The Molecular
Probes.RTM. Handbook" (www.invitrogen.com) for a description of
various uses for the dyes of the present invention.
[0318] The dyes may also be used to detect biological phenomena
using fluorescent resonance energy transfer FRET or bioluminescence
resonance energy transfer (BRET). See, e.g. "The Molecular
Probes.RTM. Handbook" (www.invitrogen.com) for a description of
various uses for the dyes of the present invention. In some
embodiments, the dyes disclosed herein can be used as BRET
acceptors. If a dye described herein is brought within the energy
transfer radius (e.g., typically <10 nm) of a luciferase and is
in the correct orientation, radiationless energy transfer will
occur, and the dye will emit light at its normal emission. There
are many methods known for bringing the dye close to a luciferase,
e.g., small molecules or quantum dots (Xia, Z and Rao, J. 2009.
Curr. Opin. Biotech 20: 37-44), and these methods enable one to
learn something about a biological system of interest.
[0319] In some embodiments, a dye disclosed herein may be
conjugated to a tag such as a chloroalkane (see U.S. Pat. No.
7,867,726). This conjugation causes the dye to be strongly
associated to a protein of interest in a cell or cell lysate if
that protein is expressed as a HaloTag.RTM. fusion. Such a fusion
of a protein of interest and HaloTag protein would be covalently
labeled with the dye conjugate. There are many other methods of
associating a dye disclosed herein with a specific protein, such as
conjugating the dye to a specific antibody. Another protein of
interest in the cell or cell lysate can then be fused to a
luciferase such as a firefly luciferase or Oplophorus luciferase,
e.g., NanoLuc.TM. luciferase from Promega Corporation (see U.S.
20100281552 and U.S. Ser. No. 13/287,986). Upon addition of a
luciferin substrate, BRET would be observed if the first and second
proteins of interest interact at a defined distance (e.g.,
typically <10 nm). Such a system allows one to study the
interaction of two specific proteins under various conditions,
e.g., inside of a living cell.
[0320] In some embodiments, the use of a dye disclosed herein in
FRET or BRET can be used to ascertain the biological interaction of
any two materials of interest such as nucleic acids, lipids,
polysaccharides, antibodies, small molecules, e.g. drugs or drug
compounds, etc. In some embodiments, the interaction of a small
molecule with a protein occurs inside of a living cell. In some
embodiments, a dye disclosed herein may be conjugated to a small
molecule, e.g., a tracer, that binds to a protein of interest in
such a way that the molecule/protein interaction is not disturbed
by the dye conjugation. If the protein of interest is expressed as
a luciferase fusion as described above, the interaction of the
small molecule with the protein can be measured inside a live cell.
Such an assay may also be used to investigate the binding of a
promiscuous small molecule with only a single protein regardless of
how many other proteins the particular small molecule may bind.
[0321] In some embodiments, the reactive dyes of the present
invention may be used to label a protein(s) or peptide(s) for
quantification of protein interactions in situ. In some aspects, a
dye label could be attached to the target protein or peptide of
interest using the reactive cyanobenzothiazole (CBT) labeling
chemistry (see U.S. Patent Application No. 2009/0263843, which is
incorporated by reference herein). The CBT labeling chemistry
requires a free, N-terminal cysteine residue on the target protein
or peptide, which can be generated in situ or in a biochemical
format, e.g., by site-specific proteolytic cleavage (e.g. cleavage
of an N-terminal reporter such as HaloTag from a C-terminal target
protein or peptide). Once proteolysis has occurred and an
N-terminal cysteine residue generated, the reactive CBT labeling
method can be used to generate a single dye label on the target
protein or peptide of interest. This method could also be used for
site-specific labeling of receptor ligands (e.g. cytokines or
peptide ligands). When the dye-labeled ligand is bound in close
proximity to a cell surface receptor labeled with a suitable energy
donor (e.g. luciferase for bioluminescence resonance energy
transfer (BRET) or a short-wavelength fluorophore for Forster
resonance energy transfer (FRET)), energy transfer can occur
between the excited state donor and fluorescent dye acceptor,
leading to an increase in emission from the conjugated dye. Energy
transfer could then be used to quantify the interaction of the
protein/peptide that has been labeled with the CBT-dye with the
donor-labeled receptor of interest. This labeling method may be
compatible with purified components, e.g., purified protein or
peptide, or in more complex samples including whole cells or cell
lysates. Furthermore, this labeling method may be useful for
testing the affinity of unlabeled proteins/peptides for a receptor
of interest by competitive displacement of the ligand-receptor
complex generating the BRET signal.
Kits
[0322] One aspect of the invention is the formulation of kits that
facilitate the practice of various assays using any of the dyes of
the invention, as described above. The kits of the invention
typically comprise a colored or fluorescent dye of the invention,
either present as a chemically reactive label useful for preparing
dye-conjugates or present as a dye-conjugate where the conjugated
substance is a specific binding pair member, or, for instance, a
nucleoside, nucleotide, oligonucleotide, polynucleotide, peptide,
protein or small molecule, e.g. drug or drug compound. The kit
optionally further comprises one or more buffering agents,
typically present as an aqueous solution. The kits of the invention
optionally further comprise additional detection reagents, a
purification medium for purifying the resulting labeled substance,
luminescence standards, enzymes, enzyme inhibitors, organic
solvent, constructs for expression of fusion proteins, e.g., fusion
proteins comprising a luciferase or HaloTag.RTM. protein fused to a
protein or target of interest, fusion proteins, or instructions for
carrying out an assay of the invention.
[0323] In some embodiments, a kit of the invention includes one or
more locus-specific primers. Instructions for use optionally may be
included. Other optional kit components may include an allelic
ladder directed to each of the specified loci, a sufficient
quantity of enzyme for amplification, amplification buffer to
facilitate the amplification, loading solution for preparation of
the amplified material for electrophoresis, genomic DNA as a
template control, a size marker to insure that materials migrate as
anticipated in the separation medium, and a protocol and manual to
educate the user and to limit error in use. The amounts of the
various reagents in the kits also can be varied depending upon a
number of factors, such as the optimum sensitivity of the process.
It is within the scope of this invention to provide test kits for
use in manual applications or test kits for use with automated
detectors or analyzers.
[0324] In other embodiments, the kit also includes a
genetically-modified cell or a vector for gene fusion, e.g., fusion
comprising a luciferase or HaloTag.RTM. protein fused to a protein
or target of interest. Instructions for use optionally may be
included.
[0325] The following Examples are intended to illustrate the
invention above and should not be construed as to narrow its scope.
One skilled in the art will readily recognize that the Examples may
suggest other ways in which the present invention could be
practiced. It should be understood that variations and
modifications may be made while remaining within the scope of the
invention.
EXAMPLES
Example 1
7-methoxytetralone oxime
##STR00030##
[0327] To a solution of 7-methoxytetralone (15 g) in MeOH (175 mL),
NH.sub.2OH (50% in H.sub.2O, 15.7 mL) and AcOH (4 mL) was added.
After stirring for 1 hour, the solution was concentrated until a
solid began to appear. The reaction was poured into dilute aqueous
NaHCO.sub.3 (500 mL), and the resulting solid was filtered to
provide the title compound (17.8 g): 1H NMR (DMSO-d6) .delta. 11.08
(s, 1H), 7.35 (s, 1H), 7.08 (d, 1H), 6.84 (d, 1H), 3.71 (s, 3H),
2.62-2.49 (m, 4H), 1.77-1.64 (m, 2H).
Example 2
8-methoxy-4,5-dihydro-1H-benzo[b]azepin-2(3H)-one
##STR00031##
[0329] To a solution of 7-methoxytetralone oxime (17.8 g) in
pyridine (400 mL), p-toluenesulfonyl chloride (26.6 g) was added
portion wise. After stirring for 18 hours, the reaction was poured
into HCl (3M, 1 L), and the resulting solid was filtered to provide
the crude tosylate (32.4 g). To this orange solid, EtOH (750 mL)
and NaOAc (77.0 g in 750 mL H.sub.2O) was added, and the resulting
suspension was heated to reflux. After refluxing for 16 hours, the
heat was removed, and the solution was concentrated until solid
began to appear. After cooling, filtered off the resulting white
solid to provide the title compound (11.2 g): 1H NMR (DMSO-d6)
.delta. 9.43 (s, 1H); 7.12 (d, 1H), 6.64 (dd, 1H), 6.52 (d, 1H),
3.69 (s, 3H), 2.56 (t, 2H), 2.12 (t, 2H), 2.07-1.98 (m, 2H).
Example 3
8-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine
##STR00032##
[0331] To a solution of
8-methoxy-4,5-dihydro-1H-benzo[b]azepin-2(3H)-one (2 g) in THF (100
mL), lithium aluminum hydride (0.79 g) was added, and the reaction
heated to reflux. After stirring for 1 hour, the heat was removed.
Water (6 mL) was added followed by NaOH (10%, 15 mL), the
flocculated solids removed by filtration, and the eluent
concentrated to provide the title compound (1.9 g) as a pale brown
oil: 1H NMR (DMSO-d6) .delta. 6.88 (d, 1H); 6.37 (d, 1H), 6.22 (dd,
1H), 5.14 (s, 1H), 3.62 (s, 3H), 2.90-2.85 (m, 2H), 2.57-2.53 (m,
2H), 1.67-1.59 (m, 2H), 1.52-1.45 (m, 2H).
Example 4
8-methoxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine
##STR00033##
[0333] To a solution of
8-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine (0.5 g) in
acetonitrile (25 mL), iodoethane (0.45 mL) and K.sub.2CO.sub.3 (1.2
g) was added, and the reaction heated to reflux. After stirring for
18 hours, the heat was removed, and the reaction was concentrated.
The resulting residue was partitioned between water (30 mL) and
EtOAc (25 mL), the layers separated, and the organic layer washed
with brine, dried (Na.sub.2SO.sub.4) and concentrated to provide
the title compound (0.54 g) as a clear oil: 1H NMR (DMSO-d6)
.delta. 6.93 (d, 1H); 6.39 (d, 1H), 6.33 (dd, 1H), 3.67 (s, 3H),
3.07 (q, 2H), 2.85-2.82 (m, 2H), 2.62-2.58 (m, 2H), 1.66-1.59 (m,
2H), 1.51-1.43 (m, 2H), 1.10 (t, 3H).
Example 5
8-hydroxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine
##STR00034##
[0335] To a solution of
8-methoxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine (0.54 g) in
CH.sub.2Cl.sub.2 (25 mL) cooled to -78.degree. C., BBr.sub.3 (1.24
mL) was added, and the reaction allowed to gradually warm to
-20.degree. C. After stirring for 3 hours, the reaction was
quenched with MeOH, allowed to warm to room temperature, and then
concentrated. The residue was dissolved in HCl (1M, 40 mL) and
stirred for 1 hour. This solution was brought to pH 12 with
K.sub.2CO.sub.3 (sat. aq.) and extracted with EtOAc (40 mL). The
organic layer was washed with brine, dried (Na.sub.2SO.sub.4) and
concentrated. The crude product was purified by silica gel
chromatography (gradient of EtOAc in heptane) to provide the title
compound (0.37 g) as a clear oil: 1H NMR (DMSO-d6) .delta. 8.88 (s,
1H), 6.79 (d, 1H); 6.29 (d, 1H), 6.16 (dd, 1H), 3.02 (q, 2H),
2.82-2.79 (m, 2H), 2.57-2.53 (m, 2H), 1.65-1.57 (m, 2H), 1.48-1.42
(m, 2H), 1.09 (t, 3H); MS expected 192 (C.sub.12H.sub.18NO, M+1),
found 192.
Example 6
Bis(ethylazepino)tetrachlororhodamine (PBI 3737)
##STR00035##
[0337] A mixture of
8-hydroxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine (50 mg),
tetrachlorophthalic anhydride (52 mg) and ZnCl.sub.2 (36 mg) was
heated to approximately 250.degree. C. for 2 minutes. The residue
was suspended in CH.sub.2Cl.sub.2/MeOH (1/1, 20 mL), filtered and
concentrated. The crude dye was purified by preparative HPLC
(gradient of ACN in 0.1% TFA in H.sub.2O) to provide the title
compound (1 mg) as a blue solid: MS expected 632
(C.sub.32H.sub.30Cl.sub.4N.sub.2O.sub.3.sup.+, M.sup.+), found 632;
.lamda.maxAbs=595 nm (MeOH), .lamda.maxEm=619 nm (MeOH).
Example 7
Synthesis of Additional Compounds
[0338] The following compounds were synthesized in the same manner
as PBI 3737 using the appropriate phenol and phthalic anhydride. In
some cases, the rosamine resulting from decarboxylation of the
rhodamine was also isolated.
TABLE-US-00001 PBI Structure Number MS .lamda..sub.maxAbs
.lamda..sub.maxEm ##STR00036## 3738 656 608 629 ##STR00037## 3761
613 610 633 ##STR00038## 3739 562 583 604 ##STR00039## 3740 562 585
611 ##STR00040## 3736 661 594 621 ##STR00041## 3760 617 596 626
##STR00042## 3762 631 601 621 ##STR00043## 3763 631 602 621
##STR00044## 3768 590 607 629 ##STR00045## 631 ##STR00046## 3970
631 604 617
Example 8
11-methoxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline
##STR00047##
[0340] To a solution of
8-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine (0.25 g) in
bromochloropropane (2 mL), Na.sub.2CO.sub.3 (0.6 g) was added, and
the reaction heated to reflux.
[0341] After stirring for 18 hours, the heat was removed, the
reaction partitioned between water (30 mL) and ether (25 mL), the
layers separated, and the organic layer washed with brine, dried
(Na.sub.2SO.sub.4) and concentrated. The crude product was purified
by silica gel chromatography (gradient of EtOAc in heptane) to
provide the title compound (0.27 g) as a clear oil: 1H NMR
(DMSO-d6) .delta. 6.82 (d, 1H); 6.36 (d, 1H), 3.68 (s, 3H),
3.04-3.00 (m, 2H), 2.89-2.85 (m, 2H), 2.60-2.57 (m, 2H), 2.53-2.49
(m, 2H), 1.70-1.61 (m, 4H), 1.48-1.40 (m, 2H); MS expected 218
(C.sub.14H.sub.20NO, M+1), found 218.
Example 9
11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline
##STR00048##
[0343] The title compound was synthesized in a similar manner as
8-hydroxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine from
11-methoxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline: 1H
NMR (DMSO-d6) .delta. 8.82 (s, 1H), 6.64 (d, 1H); 6.22 (d, 1H),
3.03-2.99 (m, 2H), 2.87-2.83 (m, 2H), 2.55-2.50 (m, 2H), 2.53-2.49
(m, 2H), 1.68-1.61 (m, 4H), 1.45-1.37 (m, 2H); MS expected 204
(C.sub.13H.sub.18NO, M+1), found 204.
Example 10
8-methoxy-5-methyl-2,3-dihydro-1H-benzo[b] azepine
##STR00049##
[0345] The title compound was synthesized following the procedure
for synthesizing 8-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine
using 8-methoxy-5-methyl-1H-benzo[b]azepin-2(3H)-one (Aust. J.
Chem. 1978, 31, 2031-2037) as starting material: 1H NMR
(DMSO-d.sub.6) .delta. 7.28 (d, 1H), 6.46 (dd, 1H); 6.28 (d, 1H),
5.89 (t, 1H), 5.30 (s, 1H), 3.77 (s, 3H), 3.40 (t, 2H), 2.41 (q,
2H), 2.14 (d, 3H); MS expected 190 (C.sub.12H.sub.16NO, M+1), found
190.
Example 11
8-hydroxy-5-methyl-2,3-dihydro-1-ethylbenzo[b]azepine
##STR00050##
[0347] The title compound was synthesized from
8-methoxy-5-methyl-2,3-dihydro-1H-benzo[b]azepine following the
alkylation procedure for
8-methoxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine followed by the
demethylation procedure used to synthesize
8-hydroxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine: .sup.1H NMR
(DMSO-d6) .delta. 9.15 (s, 1H), 7.05 (d, 1H), 6.31-6.25 (m, 2H),
5.80 (t, 1H), 5.30 (s, 1H), 3.12-3.03 (m, 4H), 2.17 (q, 2H), 1.97
(s, 3H), 1.09 (t, 3H); MS expected 203 (C.sub.13H.sub.18NO, M+1),
found 203.
Example 12
8-hydroxy-5-methyl-2,3,4,5-tetrahydro-1-ethylbenzo[b] azepine
##STR00051##
[0349] A suspension of
8-hydroxy-5-methyl-2,3-dihydro-1-ethylbenzo[b]azepine (0.14 g) and
Pd/C (10 mg) in MeOH (10 mL) was purged with H.sub.2 and then
stirred under 1 atm H.sub.2 for 3 hours. The reaction was then
filtered over Celite, the eluent concentrated, and the crude
reaction purified over silica gel (gradient of EtOAc in heptane) to
provide the title compound (0.09 g) as a clear oil: 1H NMR
(CDCl.sub.3) .delta. 7.26 (s, 1H), 6.97 (d, 1H), 6.46-6.33 (m, 2H),
3.23-3.02 (m, 4H), 2.79-2.67 (m, 2H), 1.81-1.53 (m, 3H), 1.29 (d,
3H), 1.19 (t, 3H); MS expected 206 (C.sub.13H.sub.20NO, M+1), found
206.
Example 13
9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline
##STR00052##
[0351] The title compound was synthesized in a similar manner as
11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinolone
starting from 5-methoxytetralone: 1H NMR (DMSO-d6) .delta. 8.69 (s,
1H), 6.55 (d, 1H); 6.27 (d, 1H), 3.05-3.02 (m, 2H), 2.92-2.89 (m,
2H), 2.68-2.64 (m, 2H), 2.54 (t, 2H), 1.69-1.60 (m, 4H), 1.45-1.37
(m, 2H); MS expected 204 (C.sub.13H.sub.18NO, M+1), found 204.
Example 14
2,3,4,5-tetrachloro-6-(9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij-
]quinoline-10-carbonyl)benzoic Acid
##STR00053##
[0353] To a solution of
9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinolone (30
mg) in dichlorobenzene (1 mL), tetrachlorophthalic anhydride (0.13
mL) was added. After stirring at reflux for 2 hours, the solvent
was removed, and the resulting crude product purified by
preparative HPLC (gradient of ACN in 0.1% TFA in H.sub.2O) to
provide the title compound (20 mg) as a green solid: MS expected
490 (C.sub.21H.sub.18Cl.sub.4NO.sub.4.sup.+, M+), found 490.
Example 15
Synthesis of Additional Compounds
[0354] The following compounds were synthesized in the same manner
as
2,3,4,5-tetrachloro-6-(9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-i-
j]quinoline-10-carbonyl)benzoic acid using the appropriate phenol
and phthalic anhydride:
TABLE-US-00002 Structure MS ##STR00054## 489 ##STR00055## 396
##STR00056## 396 ##STR00057## 464 ##STR00058## 464 ##STR00059##
424
Example 16
Bis(azepinopiperidino)-tetrachlororhodamine (PBI 3861)
##STR00060##
[0356] To a solution of
2,3,4,5-tetrachloro-6-(9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-i-
j]quinoline-10-carbonyl)benzoic acid (20 mg) and
9-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinolone (14
mg) in DMF (1 mL), trimethylsilylpolyphosphate (0.25 mL) was added.
After stirring at 80.degree. C. for 1 hour, water (1 mL) was added,
and the resulting solution purified by preparative HPLC (gradient
of ACN in 0.1% TFA in H.sub.2O) to provide the title compound (10
mg) as a blue solid: MS expected 656
(C.sub.34H.sub.30Cl.sub.4N.sub.2O.sub.3.sup.+, M+), found 656;
.lamda.maxAbs=605 nm (MeOH), .lamda.maxEm=620 nm (MeOH).
Example 17
Synthesis of Additional Compounds
[0357] The following compounds were synthesized in the same manner
as bis(azepinopiperidino)-tetrachlororhodamine using the
appropriate phenol and ketophenol from Example 14 or 15.
TABLE-US-00003 PBI Structure Number MS .lamda..sub.maxAbs
.lamda..sub.maxEm ##STR00061## 4273 694 647 713 ##STR00062## 4302
693 668 741 ##STR00063## 4335 641 632 690 ##STR00064## 4351 548 602
662 ##STR00065## 4352 670 614 632 ##STR00066## 4379 591 590 612
##STR00067## 4382 497 564 596 ##STR00068## 4428 566 583 604
##STR00069## 4464 641 611 644 ##STR00070## 4484 667 614 646
##STR00071## 4490 617 592 616 ##STR00072## 4496 643 601 621
##STR00073## 4497 616 598 637 ##STR00074## 4553 682 619 637
##STR00075## 575 602 642 ##STR00076## 617 617 638 ##STR00077## 4624
497 571 601
Example 18
Bis(piperidineazepino)-trichlororhodamine Mercaptoacetic Acid (PBI
3769)
##STR00078##
[0359] To a solution of bis(piperidineazepino)-tetrachlororhodamine
(PBI 3738, 0.60 g) and diisopropylethylamine (0.32 mL) in DMF (10
mL), mercaptoacetic acid (0.13 mL) was added. After stirring for 2
hours, the resulting product was purified by preparative HPLC
(gradient of ACN in 0.1% TFA in H.sub.2O) to provide the title
compound (0.35 g) as a blue solid: MS expected 712
(C.sub.36H.sub.33Cl.sub.3N.sub.2O.sub.5S.sup.+, M+), found 712;
.lamda.maxAbs=606 nm (MeOH), .lamda.maxEm=627 nm (MeOH).
Example 19
Synthesis of Additional Compounds
[0360] The following compounds were synthesized in the same manner
as PBI 3769 using the appropriate halorhodamine:
TABLE-US-00004 PBI Structure Number MS .lamda..sub.maxAbs
.lamda..sub.maxEm ##STR00079## 662 610 633 ##STR00080## 3865 637
617 642 ##STR00081## 749 ##STR00082## 647 589 613 ##STR00083## 4577
699 600 620 ##STR00084## 4555 727 610 629 ##STR00085## 4559 647 604
645 ##STR00086## 4568 689 613 633 ##STR00087## 4681 721 614 652
Example 20
Bis(piperidinoazepino)-pentafluororosamine (PBI 3860)
##STR00088##
[0362] A solution of pentafluorobenzaldehyde (40 mg) and
11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinolone (50
mg) in H.sub.2SO.sub.4 (60% aqueous, 2 mL) was stirred at
150.degree. C. for 10 minutes. The resulting solution was purified
by preparative HPLC (gradient of ACN in 0.1% TFA in H.sub.2O) to
provide the title compound (20 mg) as a blue solid: MS expected 565
(C.sub.33H.sub.30F.sub.5N.sub.2O.sup.+, M+), found 565;
.lamda..sub.maxAbs=620 nm (MeOH), .lamda..sub.maxEm=642 nm
(MeOH).
Example 21
Bis(piperidineazepino)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-carbamoy-
l)rhodamine (PBI 3781)
##STR00089##
[0364] To a solution of bis(piperidineazepino)-6-carboxyrhodamine
(10 mg) and diisopropylethylamine (0.02 mL) in DMF (1 mL), TSTU (8
mg) was added. After stirring for 15 minutes,
2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine HCl (7 mg) was added,
which was synthesized according to the procedure described in H.
Benink, M. McDougall, D. Klaubert, G. Los, BioTechniques 2009, 47,
769-774 (which is incorporated by reference herein). After stirring
another 30 minutes, the reaction mixture was purified by
preparative HPLC (gradient of ACN in 0.1% TFA in H.sub.2O) to
provide the title compound (1 mg) as a blue solid: MS expected 769
(C.sub.45H.sub.55ClN.sub.3O.sub.6.sup.+, M+), found 769.
Example 22
Synthesis of Additional Compounds
[0365] The following compounds were synthesized in the same manner
as PBI 3781 using the appropriate dye carboxylic acid and
amine:
TABLE-US-00005 PBI Structure Number MS ##STR00090## 3780 868
##STR00091## 3782 838 ##STR00092## 3783 917 ##STR00093## 3905 926
##STR00094## 3906 838 ##STR00095## 1280 ##STR00096## 3954 838
##STR00097## 4356 953 ##STR00098## 4357 1042 ##STR00099## 4830 769
##STR00100## 4839 857 ##STR00101## 4840 945
Example 23
Bis(piperidineazepino)trichlororhodamine acetoallylaminodU 5'-DMT
3'-phosphoramidite (PBI 3885)
##STR00102##
[0367] Solid bis(piperidineazepino)trichlororhodamine
acetoallylaminodU 5'-DMT (1.0 g) was flushed with N.sub.2 and
dissolved in dry CH.sub.2Cl.sub.2 (10 mL). To this solution,
5-ethylthiotetrazole (30 mg) followed by
2-cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite (0.31 mL)
was added. After stirring for 90 minutes, the reaction mixture was
added to heptane dropwise. The slurry was stirred for 5 minutes and
filtered to provide the title compound (1.0 g) as a blue solid: MS
expected 1480 (C.sub.78H.sub.84Cl.sub.3N.sub.7O.sub.12PS+, M+),
found 1480.
Example 24
Bis(piperidineazepino)-trichlororhodamine Mercaptoacetic Acid SE
(PBI 4574)
##STR00103##
[0369] To a solution of bis(piperidineazepino)-trichlororhodamine
mercaptoacetic acid (10 mg) and diisopropylethylamine (0.02 mL) in
CH.sub.2Cl.sub.2 (0.5 mL), 2-succimido-1,1,3,3-tetramethyluronium
tetrafluoroborate (TSTU) (8 mg) was added. After stirring for 1
hour, the reaction mixture was poured into monosodium citrate (250
mM, 15 mL), extracted with CH.sub.2Cl.sub.2 (10 mL) three times,
and the combined organic layers dried (Na.sub.2SO4) and
concentrated to provide the title compound (5 mg) as a blue solid:
MS expected 809 (C.sub.40H.sub.36Cl.sub.3N.sub.3O.sub.7S.sup.+,
M+), found 809.
Example 25
Bis(piperidineazepino)-3,5-bissulforhodamine (PBI 3904)
##STR00104##
[0371] A mixture of
11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinoline (50
mg) and 4-formylbenzene-1,3-disulfonic acid disodium salt hydrate
(38 mg) in 1 mL of concentrated sulfuric acid was heated with
stirring in an open vessel to 100.degree. C. by means of an oil
bath for five hours. After this time, the reaction was removed from
the oil bath, and ice was slowly added while stirring until
liquefied. The acidified water was then decanted from an oily
residue which was further washed 2 more times with water. The
residue was then dissolved in methanol, and the solvent evaporated
depositing the residue onto celite. The crude product was purified
by silica gel chromatography (gradient of MeOH in CH.sub.2Cl.sub.2)
to provide the title compound as a red solid (78 mg): MS expected
635 (C.sub.33H.sub.35N.sub.2O.sub.7S.sub.2, M+), found 635;
.lamda.maxAbs=590 nm (MeOH), .lamda.maxEm=613 nm (MeOH).
Example 26
Bis(piperidineazepino)-5-sulfonylchloride sulforhodamine
[0372] A solution of bis(piperidineazepino)-3,5-bissulforhodamine
(PBI 3904, 70 mg) in POCl.sub.3 (2 mL) and THF (2 mL) was stirred
for 1 hour and then concentrated under reduced pressure. After
stirring another 30 minutes, the reaction mixture was purified by
preparative HPLC (gradient of ACN in 0.1% TFA in H.sub.2O) to
provide the title compound (1 mg) as a blue solid: MS expected 769
(C.sub.45H.sub.55ClN.sub.3O.sub.6.sup.+, M+), found 769.
Example 27
Bis(piperidineazepino)-5-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)sulfonyl)
sulforhodamine (PBI 3909)
##STR00105##
[0374] A solution of bis(piperidineazepino)-3,5-bissulforhodamine
(PBI 3904, 70 mg) in POCl.sub.3 (2 mL) and THF (2 mL) was stirred
for 1 hour and then concentrated under reduced pressure. This crude
sulfonyl chloride was dissolved in CH.sub.2Cl.sub.2 (5 mL), and
triethylamine (0.23 mL) and
2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine HCl (43 mg) were added.
After stirring for 3 days, the reaction mixture was concentrated.
The crude product was dissolved in DMF and purified by preparative
HPLC (gradient of ACN in 0.1% TFA in H.sub.2O) to provide the title
compound (6 mg) as a blue solid: MS expected 840
(C.sub.43H.sub.54ClN.sub.3O.sub.8S.sub.2.sup.+, M+), found 840.
Example 28
5-methoxy-2-aminonaphthalene
##STR00106##
[0376] To a solution of 6-aminonaphth-1-ol (1.0 g) in DMF (50 mL),
NaH (60% in mineral oil, 0.17 g) was added, and the reaction was
stirred for 1 hour. Iodomethane (0.39 mL) was added, and the
reaction was stirred for another 1 hour. The reaction was then
partitioned between NaHCO.sub.3 (aq., 150 mL) and EtOAc (100 mL),
the layers separated, and the organic layer washed with brine,
dried (Na.sub.2SO.sub.4) and concentrated. The crude reaction was
purified over silica gel (gradient of EtOAc in heptane) to provide
the title compound (0.8 g) as an orange oil:: 1H NMR (DMSO-d6)
.delta. 7.81 (d, 1H); 7.16 (dd, 1H), 7.04 (d, 1H), 6.85 (dd, 1H),
6.75 (d, 1H), 6.53 (dd, 1H), 5.32 (s, 2H), 3.86 (s, 3H).
Example 29
5-hydroxy-2-(dimethylamino)naphthalene
##STR00107##
[0378] From the purification of 5-methoxy-2-aminonaphthalene
5-methoxy-2-(dimethylamino)naphthalene was also isolated. This
compound was demethylated in the same manner as
8-hydroxy-2,3,4,5-tetrahydro-1-ethylbenzo[b]azepine to give the
title compound: 1H NMR (DMSO-d6) .delta. 9.72 (s, 1H), 7.93 (d,
1H); 7.10-7.05 (m, 3H), 6.82 (d, 1H), 6.52 (dd, 1H), 2.96 (s, 6H);
MS expected 188 (C.sub.12H.sub.13NO, M+1), found 188.
Example 30
9-hydroxy-1,2,3,5,6,7-hexahydrobenzo[f]pyrido[3,2,1-ij]quinolone
##STR00108##
[0380] The title compound was synthesized in a similar manner as
11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinolone
starting from 5-methoxy-2-aminonaphthalene: 1H NMR (DMSO-d6)
.delta. 9.55 (s, 1H), 7.55 (s, 1H); 7.08-7.00 (m, 2H), 6.48 (dd,
1H), 3.14 (q, 4H), 2.86 (q, 2H), 2.02-1.86 (m, 4H); MS expected 240
(C.sub.16H.sub.18NO, M+1), found 240.
Example 31
3-methoxy-1-(dimethylamino)naphthalene
##STR00109##
[0382] To a solution of 3-methoxy-1-aminonaphthalene (U.S. Pat. No.
7,018,431 B2, 0.13 g) in DMF (5 mL), K.sub.2CO.sub.3 (0.31 g) and
iodomethane (0.09 mL) were added. After stirring for 24 hours, the
reaction was partitioned between water (30 mL) and EtOAc (30 mL),
the layers separated, and the organic layer washed with brine,
dried (Na.sub.2SO.sub.4) and concentrated. The crude reaction
purified over silica gel (gradient of EtOAc in heptane) to provide
the title compound (0.14 g) as a clear oil: 1H NMR (DMSO-d.sub.6)
.delta. 7.99 (d, 1H); 7.74 (d, 1H), 7.40 (ddd, 1H), 7.30 (ddd, 1H),
6.95 (d, 1H), 6.67 (d, 1H), 3.83 (s, 3H), 2.78 (s, 6H); MS expected
202 (C.sub.13H.sub.16NO, M+1), found 202.
Example 32
3-hydroxy-1-(dimethylamino)naphthalene
##STR00110##
[0384] The title compound was synthesized in a similar manner as
11-hydroxy-2,3,5,6,7,8-hexahydro-1H-azepino[3,2,1-ij]quinolone
starting from 3-methoxy-1-(dimethylamino)naphthalene: 1H NMR
(DMSO-d6) .delta. 9.53 (s, 1H), 7.95 (d, 1H); 7.60 (d, 1H), 7.32
(ddd, 1H), 7.21 (ddd, 1H), 6.75 (d, 1H), 6.64 (d, 1H), 2.77 (s,
6H).
Example 33
Bis(piperidineazepino)-6-((2-(2-(2-(2-(6-carboxyamido-2-cyanobenzothiazoly-
l)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)rhodamine (PBI 5122)
##STR00111##
[0386] To a solution of t-boc-N-amido-dPEG.RTM.4-acid (Quanta
BioDesign, 1 g) and N-methylmorpholine (0.3 mL) in THF (25 mL),
isobutyl chloroformate (0.36 mL) was added. After stirring for 30
min, 6-amino-2-cyanobenzothiazole (White et. al., J. Am. Chem. Soc.
88, 2015 (1966), 0.48 g) was added, and the reaction stirred
overnight. The reaction was then filtered, and the eluent
concentrated. The crude reaction purified over silica gel (gradient
of MeOH in CH.sub.2Cl.sub.2) to provide a clear oil (1.4 g).
[0387] The oil from the previous step was dissolved in 15%
thioanisole in trifluoroacetic acid (25 mL) at 0.degree. C. The
reaction was stirred for 3 hours, diluted with diethyl ether and
then concentrated to dryness. The reaction was then filtered, and
the eluent concentrated. The crude reaction purified over silica
gel (gradient of MeOH in CH.sub.2Cl.sub.2) and immediately carried
on to the next step.
[0388] To a solution of bis(piperidineazepino)-6-carboxyrhodamine
(76 mg) and diisopropylethylamine (0.04 mL) in CH.sub.2Cl.sub.2 (1
mL) was added TSTU (8 mg). After stirring for 15 min, the crude
amine from the previous step (62 mg) was added. After stirring
another 30 min, the reaction mixture was purified by preparative
HPLC (gradient of ACN in 0.1% TFA in H2O) to provide the title
compound (6 mg) as a blue solid: MS expected 967 (C54H59N6O9S+,
M+1), found 967.
Example 34
General Procedures for Labeling Oligonucleotides with the Dyes of
the Present Invention
[0389] A. Oligonucleotide labeling with N-Hydroxysuccinimidyl Ester
Dyes
[0390] i. 1 .mu.mole scale
[0391] A 5'-amino labeled oligonucleotide was synthesized on an ABI
394 DNA synthesizer (1 .mu.mmole) using 5' Amino modifier C6 TFA
amidite from Glen Research. Deprotection was performed in
concentrated ammonium hydroxide overnight at 60.degree. C. to yield
a 5'-aminohexyl labeled oligonucleotide. The resulting
oligonucleotide was evaporated to dryness, redissolved in 1 ml 0.5
M NaCl (performed for counter-ion exchange) and desalted on NAP-10
size exclusion cartridge (GE Healthcare). After desalting, the
oligonucleotide was evaporated to dryness followed by
re-dissolution in 200 .mu.l 0.5 M sodium carbonate buffer, pH 9.0.
The succinimidyl ester dyes (PBI 4451, 4510, 4574, 4563, 4566 and
4509) were dissolved in DMF at a concentration of 20 .mu.l/mg.
2.times.20 .mu.l aliquots of the dye/DMF solution were added to the
dissolved oligonucleotide at 30 minutes apart. After the second
addition of the dye/DMF solution, the reaction was mixed for 1 hour
at 20.degree. C. After one hour, it was diluted to 1 ml with water
and desalted on a NAP-10 column (GE Healthcare). The NAP-10 eluate
was purified by reversed phase HPLC on a Phenomonex Jupiter C18
column using an acetonitrile/0.1M TEAA buffer system. The HPLC
purified oligonucleotide was evaporated to dryness redissolved in
0.01M triethylammonium bicarbonate and desalted on a NAP-10 column.
After final desalt step, the oligonucleotide was evaporated to
dryness and stored at -20.degree. C.
[0392] ii. 100 .mu.mole scale
[0393] A 5'-amino labeled oligonucleotide was synthesized on an
AKTA OligoPilot (100 .mu.mole) DNA synthesizer using 5' Amino
modifier C6 TFA amidite from Glen Research. Deprotection was
performed in concentrated ammonium hydroxide overnight at
60.degree. C. to yield a 5'-aminohexyl labeled oligonucleotide. The
resulting oligonucleotide was evaporated to dryness, redissolved in
75 ml 2 M NaCl and desalted on a 500 ml G-25 column (GE
Healthcare). After desalting, the oligonucleotide was evaporated to
dryness followed by re-dissolution in 50 ml 0.5 M sodium carbonate
buffer, pH 9.0. The succinimidyl ester dyes (PBI 4451, 4510, 4574,
4563, 4566 and 4509) were dissolved in DMF at a concentration of 20
.mu.l/mg. 2400 .mu.l of the dye/DMF solution was added drop wise to
the dissolved oligonucleotide. The reaction was mixed for 1 hour at
room temperature. The dye conjugated oligonucleotide was
neutralized with sodium acetate, pH 5.5, solution and precipitated
from 2.times. volume of ethanol. The precipitated oligonucleotide
was centrifuged at 9000 rpm for 60 minutes, the supernatant
decanted to waste, and the resulting solid dissolved in 70 ml water
and purified by ion-exchange chromatography. The oligonucleotide
was concentrated and desalted using tangential flow ultrafiltration
and subsequently evaporated to dryness. It was stored at
-20.degree. C.
B. Oligonucleotide labeling with Phosphoramidites Dyes
[0394] i. 1 .mu.mole scale
[0395] A 5'-labeled oligonucleotide was synthesized on an ABI 394
DNA synthesizer (1 mmole) using the phosphoramidite dye (PBI 3885)
of the present invention dissolved to 0.1M in acetonitrile.
Deprotection was performed in t-butylamine/MeOH/water (25/25/50)
overnight at 60.degree. C. to yield a 5'-labeled oligonucleotide.
The resulting oligonucleotide was evaporated to dryness,
redissolved in 0.01 M triethylammonium bicarbonate and purified by
reversed phase HPLC on a Phenomonex Jupiter C18 column using an
acetonitrile/0.1 M TEAA buffer system. The HPLC purified
oligonucleotide was evaporated to dryness, redissolved in 0.01 M
triethylammonium bicarbonate and desalted on a NAP-10 column (GE
Healthcare). After final desalt step, the oligonucleotide was
evaporated to dryness and stored at -20C.
[0396] ii. 100 .mu.mole scale
[0397] A 5'-labeled oligonucleotide was synthesized on an AKTA
OligoPilot DNA synthesizer (100 .mu.mole) using the phosphoramidite
dye (PBI 3885) of the present invention dissolved to 0.1 M in
acetonitrile. Deprotection was performed in t-butylamine/MeOH/water
(25/25/50) overnight at 60.degree. C. to yield a 5'-labeled
oligonucleotide. The resulting oligonucleotide was evaporated to
dryness, redissolved in 0.01M triethylammonium bicarbonate and
purified by ion-exchange HPLC. The resulting purified
oligonucleotide was concentrated and desalted using tangential flow
ultrafiltration and evaporated to dryness. The labeled
oligonucleotide was stored at -20.degree. C.
Example 35
PCR and Multiplex PCR Using Oligonucleotides Labeled with the Dyes
of The Present Invention
[0398] To demonstrate the ability to perform a 6-dye multiplex PCR
with the dyes of the present invention, multiplex reactions were
performed containing primer pairs for 24 STR loci.
[0399] For the multiplex reactions, a 5.times. primer pair master
mix for 21 STR loci ("5.times. 21-STR Primer Mix") as outlined in
Table 1 and a 5.times. reaction master mix ("5.times. Reaction
Master Mix") containing reaction buffer and GoTaq.RTM. Hot Start
DNA polymerase were made. Also, in addition to the 21 STR loci
primer pairs, additional primer pairs were prepared as described in
Table 2 using dyes of the present invention. These additional
primer pairs were made to 150 .mu.M in 1 mM MOPS with 0.1 mM EDTA
with final .about.pH 7.5 at 25.degree. C.
TABLE-US-00006 TABLE 1 Primer Pair Concentration Locus Dye (uM in
5.times.) Amelogenin 5FAM 2.88 D3S1358 5FAM 0.88 D1S1656 5FAM 1.42
D6S1043 5FAM 1.32 D13S317 5FAM 1.6 Penta E 5FAM 7.88 Penta D JOE
3.68 D16S539 JOE 2.4 D18S51 JOE 1.18 D2S1338 JOE 2.04 CSF1PO JOE
1.4 TH01 ET TMR 1.38 vWA ET TMR 1.8 D21S11 ET TMR 1.55 D7S820 ET
TMR 2.4 D5S818 ET TMR 2.02 TPDX ET TMR 1.89 D8S1179 ET ROX 1.7
D12S391 ET ROX 3.75 D19S433 ET ROX 1.05 FGA ET ROX 1.28
TABLE-US-00007 TABLE 2 Locus Dye D22S1045 6FAM ET PBI 4510 (624 nm)
D2S441 6FAM ET PBI 4510 (624 nm) DYS391 6FAM ET PBI 4510 (624 nm)
D22S1045 6FAM ET PBI 4563 (640 nm) D2S441 6FAM ET PBI 4563 (640nm)
DYS391 6FAM ET PBI 4563 (640 nm) D22S1045 6FAM ET PBI 4574 (634 nm)
D2S441 6FAM ET PBI 4574 (634 nm) DYS391 6FAM ET PBI 4574 (634
nm)
[0400] Multiplex reactions were then set up as follows:
[0401] A. Multiplex 1 Mix (for 10 reactions): [0402] 5.times.
2l-STR Primer Mix: 50 .mu.l [0403] 5.times. Reaction Master Mix: 50
.mu.l [0404] 4510-D22S1045 primer pair (0.6 .mu.M): 1 .mu.l [0405]
4510-D2S441 primer pair (0.6 .mu.M): 1 .mu.l [0406] 4510-DYS391
primer pair (0.6 .mu.M): 1 .mu.l [0407] Nuclease free water: 137
.mu.l
[0408] B. Multiplex 2 Mix (for 10 reactions): [0409] 5.times.21-STR
Primer Mix: 50 .mu.l [0410] 5.times. Reaction Master Mix: 50 .mu.l
[0411] 4563-D22S1045 primer pair (0.6 .mu.M): 1 .mu.l [0412]
4563-D2S441 primer pair (0.6 .mu.M): 1 .mu.l [0413] 4563-DYS391
primer pair (0.6 .mu.M): 1 .mu.l [0414] Nuclease free water: 137
.mu.l
[0415] C. Multiplex 3 Mix (for 10 reactions): [0416] 5.times.
21-STR Primer Mix: 50 .mu.l [0417] 5.times. Reaction Master Mix: 50
.mu.l [0418] 4574-D22S1045 primer pair (0.6 .mu.M): 1 .mu.l [0419]
4574-D2S441 primer pair (0.6 .mu.M): 1 .mu.l [0420] 4574-DYS391
primer pair (0.6 .mu.M): 1 .mu.l [0421] Nuclease free water: 137
.mu.l
[0422] D. Multiplex 4 Mix (for 10 reactions): [0423] 5.times.21-STR
Primer Mix: 50 .mu.l [0424] 5.times. Reaction Master Mix: 50 .mu.l
[0425] 4510-D22S1045 primer pair (2.4 .mu.M): 4 .mu.l [0426]
4510-D2S441 primer pair (2.4 .mu.M): 4 .mu.l [0427] 4510-DYS391
primer pair (2.4 .mu.M): 4 .mu.l [0428] Nuclease free water: 128
.mu.l
[0429] E. Multiplex 5 Mix (for 10 reactions): [0430] 5.times.
21-STR Primer Mix: 50 .mu.l [0431] 5.times. Reaction Master Mix: 50
.mu.l [0432] 4563-D22S1045 primer pair (2.4 .mu.M): 4 .mu.l [0433]
4563-D2S441 primer pair (2.4 .mu.M): 4 .mu.l [0434] 4563-DYS391
primer pair (2.4 .mu.M): 4 .mu.l [0435] Nuclease free water: 128
.mu.l
[0436] F. Multiplex 3 Mix (for 10 reactions): [0437] 5.times.
21-STR Primer Mix: 50 .mu.l [0438] 5.times. Reaction Master Mix: 50
.mu.l [0439] 4574-D22S1045 primer pair (2.4 .mu.M): 4 .mu.l [0440]
4574-D2S441 primer pair (2.4 .mu.M): 4 .mu.l [0441] 4574-DYS391
primer pair (2.4 .mu.M): 4 .mu.l [0442] Nuclease free water: 128
.mu.l
[0443] 24 .mu.l of each multiplex mix was then added to a well of a
96-well PCR plate. 1 .mu.l of 1 ng/.mu.L male DNA (2800M Promega
Cat. # DD7101 or 9948 Promega Cat. # DD206A) or 0.5 ng/.mu.L male
DNA (C274 or QC2; Promega) was added to each well. Various
single-source male DNA samples were used to determine variability
in balance and bleedthrough/bridging of the dyes with various
allele patterns. Reactions were then run on an Applied Biosystems
9700 thermal cycler using the following cycling conditions:
96.degree. C. for 1 minutes; then 30 cycles of 94.degree. C. for 10
seconds, 59.degree. C. for 1 minutes and 72.degree. C. for 30
seconds; 60.degree. C. for 10 minutes; and a 4.degree. C. soak.
Reactions were then analyzed on an Applied Biosystems 3500xL
Genetic Analyzer (FIGS. 4-8).
[0444] This example demonstrates that the dyes of the present
invention work very well with respectable signals and very little
bleedthrough or bridging in multiplex PCR.
Example 36
Cell Labeling Using the Dyes of the Present Invention
[0445] To determine the ability of the dyes of the present
invention to be used for labeling in cells, the dyes were
conjugated to a HaloTag.RTM. ligand (Promega) to monitor
activity/movement of the HaloTag.RTM. protein or HaloTag.RTM.
fusion protein. For cell labeling, the ligands #3780, 3781, 3782,
3783, 3905, 3906, 3954, 4356 and 4357 (Table 3) were used. U2OS
cells stably expressing HaloTag.RTM. protein in the nucleus
(HT-NLS) were used to test for the cell permeability of the
ligands. In some cases, the efficiency of the removal of unbound
ligand was also determined. Ligands for which HT-NLS imaging did
not show obvious ligand removal issues were further tested in cells
stably expressing HaloTag.RTM. protein in their cytoplasm using a
p65-HaloTag.RTM. fusion (p65-HT) and U2OS cells not expressing
HaloTag.RTM. protein. The U2OS p65-HT stable cells were used to
establish the imaging parameters for a medium to low expressing
fusion protein. These same parameters were then used to assess
removal of unbound ligand in U2OS cells not expressing HaloTag. All
ligands were diluted in DMSO to 10 mM prior to use with cells.
TABLE-US-00008 TABLE 3 Ligand Number Ligand Structure Dye Number
3780 ##STR00112## 3781 ##STR00113## PBI 3739 3782 ##STR00114## PBI
3762 3783 ##STR00115## PBI 3769 3905 ##STR00116## PBI 3762 3906
##STR00117## PBI 3763 3954 ##STR00118## PBI 3954 4356 ##STR00119##
4357 ##STR00120##
[0446] For all imaging experiments, U2OS cells were plated in
Lab-Tek II CG (Nunc) chambered coverslips and left overnight at
37.degree. C.+5% CO.sub.2 to attach. Cells were exposed to 1 .mu.M
ligand by a rapid label protocol. Briefly, cells were exposed to
the ligand for 15 minutes in the presence of ATCC-recommended
complete media at 37.degree. C.+5% CO.sub.2 and 800 .mu.g/ml G418
(Promega). After labeling, cells were rinsed 3 times with complete
media and incubated for 30 minutes at 37.degree. C.+5% CO.sub.2.
The media was then replaced with fresh complete media, and cells
transferred to a confocal microscope for imaging.
[0447] In some cases, U2OS cells stably expressing HT-NLS were
labeled by a no-wash protocol. Briefly, cells were exposed to 100
nM ligand overnight at 37.degree. C.+5% CO.sub.2. In these cases,
the ligand was added at the time of cell plating. On the following
day, the ligand containing media was replaced with fresh complete
media, and cells were transferred to a confocal microscope for
imaging.
[0448] Confocal images were acquired using an Olympus Fluoview
FV500 confocal microscope (Olympus, USA) outfitted with a
37.degree. C.+CO.sub.2 environmental chamber (Solent Scientific
Ltd., UK) and appropriate filter sets. See FIGS. 9-16.
[0449] In order to quantify labeling efficiency of the ligands,
SDS-PAGE analysis was performed. Briefly, cells were plated as
above for imaging and were first labeled with 1 .mu.M of ligand for
15 minutes at 37.degree. C.+5% CO.sub.2. The ligand-containing
media was then replaced with media containing 5 .mu.M HaloTag.RTM.
TMR ligand (Promega; Cat. No. G8252) and incubated for 15 minutes
at 37.degree. C.+5% CO.sub.2. Cells were then rinsed 3 times and
washed for 30 minutes at 37.degree. C.+5% CO.sub.2. The cells were
then rinsed once with 1.times.PBS, collected in 1.times.SDS gel
loading buffer (4.times. buffer (0.24M Tris, 2% SDS, 50.4%
Glycerol, 0.4M DTT, 3 mM Bromophenol Blue and Hydrochloric Acid to
pH6.8) diluted in water), placed at 95.degree. C. for 5 minutes and
loaded on a 4-20% Tris-Glycine precast gel (Invitrogen). The gel
was then scanned using a Typhoon 9410 (Amersham Biosciences) (FIG.
17).
[0450] To determine cell viability after labeling, cells were
plated in white tissue culture treated Costar 96-well plates
(Fisher Scientific). Both the CellTiter-Glo.RTM. Luminescent Cell
Viability Assay and HaloTag.RTM. protein arrays were performed as
per manufacturer protocols (Promega). Briefly, for the
CellTiter-Glo assay, 100 .mu.l of the CellTiter-Glo reagent was
added to the 100 .mu.l of media containing cells. The contents were
mixed on an orbital shaker for 2 minutes and incubated at room
temperature for 10 minutes. Luminescence was measured using a
GloMax.RTM. Multi Detection System (Promega). The luminescent
signal generated is directly proportional to the amount of ATP
present in the sample which is directly proportional to the number
of cells present in the culture.
[0451] In order to assess the use of ligand 3782 for gel based
analysis, its performance was compared to that of the HaloTag.RTM.
TMR Ligand (Promega Cat. No. G8252). To do this, the standard
Promega protocol for labeling proteins expressed in a cell-free
system was used. Briefly, each ligand was diluted to 10 .mu.M in
1.times.PBS, and 1 .mu.l of each ligand added to 2 .mu.l each of
HaloTag.RTM.-GST Standard Protein (Promega) or HaloTag.RTM.-protein
G purified from E. coli. 7 .mu.l of 1.times.PBS was then added to
each labeling reaction for a total volume of 10 .mu.l, and
reactions were incubated for 30 minutes at room temperature
protected from light. 5 .mu.l of each labeling reaction was then
added to 5 .mu.l of 2.times.SDS gel loading buffer and heated to
70.degree. C. for 2 minutes. The samples were then loaded and run
on a SDS-polyacrylamide gel and visualized using a fluorescent
scanner as described above (FIGS. 18 and 19).
Example 37
Synthesis of
5-.beta.2-(4-(3-tert-Butyl-5-(3-phenylureido)-1H-pyrazol-1-yl)benzylamino-
)-2-oxoethyl)carbamoyl)-2-bis(piperidineazepino)rhodamine
(PBI-4838)
##STR00121##
[0453] The title compound was synthesized using
bis(piperidineazepino)-6-carboxyrhodamine and
1-(1-(4-((2-aminoacetamido)methyl)phenyl)-3-tert-butyl-1H-pyrazol-5-yl)-3-
-phenylurea (Tecle et al. 2009, Chem. Biol. Drug Des., 74:547-559)
in a manner similar to PBI-3781 (Example 21): MS expected 815
(C.sub.58H.sub.61N.sub.8O.sub.6.sup.+, M+), found 815.
Example 38
Bioluminescence Resonance Energy Transfer (BRET) using PBI 4828
[0454] To demonstrate the ability of the dyes of the present
invention to be used in BRET applications, a fluorescent dye tracer
comprising a dye of the present invention, PBI 4838, was generated
to monitor binding of a known drug to a kinase target in living
cells. In this example, a p38alpha kinase inhibitor, BIRB796, was
used as a scaffold to generate a fluorescent tracer for inactive
p38alpha kinase comprising PBI 4838 (Tecle et al. 2009. Chem Biol
Drug Des: 74: 547-559; FIG. 20). The tracer was then applied to
living or lysed cells expressing a NanoLuc luciferase-p38alpha
kinase fusion protein. Upon addition of a furimazine substrate for
the NanoLuc luciferase, dose-dependent BRET was then measured in
both living and lysed cells.
[0455] HEK293 cells (20,000 cells per well in 96-well format) were
transiently transfected (Fugene HD, Promega Corporation) with pF5
plasmid DNA (Promega Corporation) encoding a NanoLuc.RTM.
luciferase-p38alpha kinase fusion protein. As a negative control,
some cells were transfected with a pF5 plasmids DNA encoding a
NanoLuc luciferase-PKC alpha fusion protein. On the second day post
transfection, the cell medium was replaced with serum-free Opti-MEM
(Life Technologies) with or without 50 ug/ml digitonin.
[0456] For tracer saturation experiments, cells were treated with
serially diluted PBI 4838 in the presence or absence of a molar
excess of BIRB796 (10 uM final).
[0457] For BIRB796 competition experiments, serially-diluted
BIRB796 was applied to cells in the presence or absence of 0.5 uM
PBI 4838 (final concentration).
[0458] Cells were then allowed to equilibrate with tracer and
BIRB796 for 2 hours at 37.degree. C. A furimazine substrate (PBI
3939), a substrate for the NanoLuc luciferase, then was added to
cells to a final concentration of 20 uM. BRET ratios were recorded
using a Varioskan luminometer at the following wavelengths: 630 nm
(acceptor)/450 nm (donor). Acceptor/donor values were used to
determine BRET ratio (FIGS. 21 and 22).
[0459] The data demonstrates that the dyes of the present invention
can be used in BRET applications. In the tracer saturation
experiments, live or permeablized cells expressing NanoLuc-p38alpha
were incubated with serially diluted PBI 4838 resulting in
dose-dependent increase in BRET. The results indicate binding of
PBI 4838 to NanoLuc fusion proteins in living cells. In the
presence of a vast molar excess of unlabeled BIRB796, the BRET
signal was inhibited completely, indicating that nearly the entire
BRET signal between NanoLuc-p38alpha and PBI 4838 is specific. For
the BIRB796 competition experiments, the data shows the ability to
competitively displace a fixed concentration of PBI-4838 in a
dose-dependent manner with unlabeled BIRB796. The control
experiments further support the specificity of the specific BRET
signal between PBI 4838 and NanoLuc p38alpha. This demonstrates the
use of cells expressing NanoLuc fused to an irrelevant kinase. In
the control experiments, cells expressing NanoLuc-PKCalpha show
only a trace amount of BRET signal in the presence of PBI 4838,
which is not affected by unlabeled BIRB796.
Example 39
Bioluminescence Resonance Energy Transfer (BRET) using PBI 3781
[0460] To demonstrate the ability of the dyes of the present
invention to be used in BRET applications, a fluorescent dye of the
present invention was conjugated to a chloroalkane as in Example
21. The dye-chloroalkane conjugate PBI 3781 covalently binds to a
HaloTag.RTM. protein or a HaloTag.RTM. fusion protein allowing
detection and/or measurement of the HaloTag or HaloTag.RTM. fusion
protein. PBI 3781 was used in combination with HaloTag.RTM. and
NanoLuc fusion proteins to measure protein-protein interactions in
living cells via BRET.
[0461] In this example, the rapamycin-mediated interaction between
a NanoLuc luciferase-FK506 binding domain of mTOR (Frb) fusion and
a HaloTag-FKBP12 fusion (FK506 binding protein) was used to
demonstrate an inducible protein-protein interaction. The
interaction was measured as a rapamycin-dependent increase of BRET
occurring between the NanoLuc-FRB fusion (donor) and HaloTag-FKBP12
fusion bound to PBI 3781 (acceptor) (FIG. 23A).
[0462] HeLa cells were co-transfected with pF5-based constructs
(Promega Corporation) for the expression of Frb-NanoLuc or
FRKBP12-HaloTag.RTM. fusion proteins using FuGENE.RTM. HD
Transfection Reagent according to the manufacturer's instructions
(Promega Corporation). The cells were then incubated overnight at
37.degree. C., 5% CO.sub.2.
[0463] One day following transfection, the cells were collected and
re-plated into wells of a white, 96-well tissue culture plate at
200,000 cells/well in 1004 DMEM+10% FBS and incubated for 24 hours
at 37.degree. C., 5% CO.sub.2.
[0464] Two days after transfection, the growth medium was replaced
with phenol red-free DMEM+5% FBS containing 500 nM of PBI 3781, and
the cells incubated for 120 minutes 37.degree. C., 5% CO.sub.2. The
cells were then treated with 504 of a serial dilution of rapamycin
in phenol-red-free DMEM and incubated for 15 minutes at room
temperature. 50 .mu.L 40 mM furimazine in phenol-red-free DMEM was
added, and BRET measured using a Thermo Varioskan plate reader (500
msec integration time; donor channel emission 450/60 bandpass
filter; acceptor channel emission 610 nm longpass color glass
filter) (FIG. 24).
[0465] This experiment demonstrates a concentration-dependent
change of absolute BRET signal following the addition of a serial
dilution of rapamycin. The experiment also demonstrates that a dye
of the present invention can be used to detect other intracellular
protein-protein interactions using suitable pairs of NanoLuc and
HaloTag.RTM. fusion proteins in combination with dye-chloroalkane
HaloTag.RTM. ligand, e.g., PBI 3781.
Example 40
Prophetic Example of Using a Dye-Cyanobenzothiazole Conjugate
[0466] The interaction of a ligand, such as epidermal growth factor
(EGF) to epidermal growth factor receptor (EGFR), can be measured
in whole cell populations. In such an example, a HaloTag-EGF fusion
protein, which contains a TEV protease cleavage site between the
HaloTag and EGF protein domains in which the last residue of TEV
protease cleavage site encodes a cysteine residue, can be used.
Upon cleavage with TEV protease, a Cys-EGF protein is generated and
may be reacted with a CBT labeled compound such as PBI 5122
(Example 33). The PBI 5122-Cys-EGF can serve as a probe capable of
binding to a NanoLuc-EGFR fusion protein expressed in living cells.
Upon binding in close proximity, energy transfer (BRET) can occur,
leading to increased dye emission. In another configuration,
unlabeled EGF, or ligands of similar binding mechanism, may disrupt
the PBI 5122-EGF: NanoLuc-EGFR complex, leading to a decrease in
energy transfer. The compatibility of these labeling chemistries
and energy transfer methods could allow for the quantification of
ligand binding events from ligands and receptors generated in whole
cells.
* * * * *